Recombinant avipox virus and method to induce an immune response

ABSTRACT

The present invention provides a method for inducing an immunological response in a mammal or avian host to a pathogen by inoculating the mammal or avian host with a synthetic recombinant avipox virus modified by the presence, in a non-essential region of the avipox genome, of DNA from any source which codes for and expresses an antigen of the pathogen. The present invention further provides a synthetic recombinant avipox virus modified by the insertion therein of DNA from any source, and particularly from a non-avipox source, into a non-essential region of the avipox genome.

This application is a continuation of application Ser. No. 07/537,890,filed Jun. 14, 1990, now U.S. Pat. No. 5,174,993 issued Dec. 29, 1992,which is a continuation of application Ser. No. 07/234,390, filed Aug.23, 1988, now abandoned, which is a continuation-in-part of applicationSer. No. 07/186,054, filed Apr. 25, 1988, now abandoned, which in turnis a continuation-in-part of application Ser. No. 07/110,335, filed Oct.20, 1987, now abandoned, which in turn is a continuation-in-part ofapplication Ser. No. 07/090,711, filed Aug. 28, 1987, now abandoned; andsaid application Ser. No. 07/537,890 is also a continuation-in-part ofapplication Ser. No. 07/090,209, filed Aug. 27, 1987, now abandoned,which is a division of application Ser. No. 06/622,135, filed Jun. 19,1984, now U.S. Pat. No. 4,722,848, issued Feb. 2, 1988, which in turn isa continuation-in-part of application Ser. No. 06/446,824, filed Dec. 8,1982, now U.S. pat. No. 4,603,112, issued Jul. 29, 1986 , which in turnis a continuation-in-part of application Ser. No. 06/334,456, filed Dec.24, 1981, now U.S. Pat. No. 4,769,330, issued Sep. 6, 1988.

The present invention relates to methods for inducing an immunologicalresponse in vertebrates, including non-avian vertebrates, usingsynthetic recombinant avipox virus. More particularly, the inventionrelates to a method for inducing an immunological response in avertebrate, particularly a mammal, to a vertebrate pathogen byinoculating the vertebrate with a synthetic recombinant avipox viruscontaining DNA which codes for and expresses the antigenic determinantsof said pathogen, and to vaccines comprising such a modified avipoxvirus. Further, the invention relates to modified avipox virus, tomethods for making and using the same, and to certain DNA sequencesproduced or involved as intermediates in the production of modifiedavipox virus and to methods for making such sequences.

BACKGROUND OF THE INVENTION

Avipox or avipoxvirus is a genus of closely related pox viruses whichinfect fowl. The genus avipox includes the species fowlpox, canary pox,junco pox, pigeon pox, quail pox, sparrow pox, starling pox, and turkeypox. The species fowlpox infects chickens, and is not to be confusedwith the human disease called chickenpox. The genus avipox shares manycharacteristics with other pox viruses and is a member of the samesubfamily, poxviruses of vertebrates, as vaccinia. Pox viruses,including vaccinia and avipox, replicate within eukaryotic host cells.These viruses are distinguished by their large size, complexity, and bythe cytoplasmic site of replication. However, vaccinia and avipox aredifferent genera and are dissimilar in their respective molecularweights, their antigenic determinants, and their host species, asreported in Intervirology Vol. 17, pages 42-44, Fourth Report of theInternational Committee on Taxonomy of Viruses (1982).

The avipox viruses do not productively infect non-avian vertebrates suchas mammals, including humans. Further, avipox does not propagate wheninoculated into mammalian (including human) cell cultures. In suchmammalian cell cultures inoculated with avipox the cells will diebecause of a cytotoxic effect, but show no evidence of productive viralinfection.

The inoculation of a non-avian vertebrate such as a mammal with liveavipox results in the formation of a lesion at the inoculation sitewhich resembles a vaccinia inoculation. However, no productive viralinfection results. Nevertheless, it has now been found that a mammal soinoculated responds immunologically to the avipox virus. This is anunexpected result.

Vaccines composed of killed pathogen or purified antigenic components ofsuch pathogens must be injected in larger quantities than live virusvaccines to produce an effective immune response. This is because livevirus inoculation is a much more efficient method of vaccination. Arelatively small inoculum can produce an effective immune responsebecause the antigen of interest is amplified during replication of thevirus. From a medical standpoint, live virus vaccines provide immunitythat is more effective and longer lasting than does inoculation with akilled pathogen or purified antigen vaccine. Thus, vaccines composed ofkilled pathogen or purified antigenic components of such pathogensrequire production of larger quantities of vaccine material than isneeded with live virus.

It is clear from the foregoing discussion that there are medical andeconomic advantages to the use of live virus vaccines. One such livevirus vaccine comprises vaccinia virus. This virus is known in the priorart to be a useful one in which to insert DNA representing the geneticsequences of antigens of mammalian pathogens by recombinant DNA methods.

Thus, methods have been developed in the prior art that permit thecreation of recombinant vaccinia viruses by the insertion of DNA fromany source (e.g. viral, prokaryotic, eukaryotic, synthetic) into anonessential region of the vaccinia genome, including DNA sequencescoding for the antigenic determinants of a pathogenic organism. Certainrecombinant vaccinia viruses created by these methods have been used toinduce specific immunity in mammals to a variety of mammalian pathogens,all as described in U.S. Pat. No. 4,603,112, incorporated herein byreference.

Unmodified vaccinia virus has a long history of relatively safe andeffective use for inoculation against smallpox. However, before theeradication of smallpox, when unmodified vaccinia was widelyadministered, there was a modest but real risk of complications in theform of generalized vaccinia infection, especially by those sufferingfrom eczema or immunosuppression. Another rare but possible complicationthat can result from vaccinia inoculation is post vaccinationencephalitis. Most of these reactions resulted from inoculatingindividuals with skin diseases such as eczema or with impaired immunesystems, or individuals in households with others who had eczema orimpaired immunological responses. Vaccinia is a live virus, and isnormally harmless to a healthy individual. However, it can betransmitted between individuals for several weeks after inoculation. Ifan individual with an impairment of the normal immune response isinfected either by inoculation or by contagious transmission from arecently inoculated individual, the consequences can be serious.

Thus, it can be appreciated that a method which confers on the art theadvantages of live virus inoculation but which reduces or eliminates thepreviously discussed problems would be a highly desirable advance overthe current state of technology. This is even more important today withthe advent of the disease known as acquired immune deficiency syndrome(AIDS). Victims of this disease suffer from severe immunologicaldysfunction and could easily be harmed by an otherwise safe live viruspreparation if they came in contact with such virus either directly orvia contact with a person recently immunized with a vaccine comprisingsuch a live virus.

OBJECTS OF THE INVENTION

It is an object of the present invention to provide a vaccine which iscapable of immunizing vertebrates against a pathogenic organism, whichhas the advantages of a live virus vaccine, and which has few or none ofthe disadvantages of either a live virus vaccine or a killed virusvaccine as enumerated above, particularly when used to immunizenon-avian vertebrates.

It is a further object of this invention to provide syntheticrecombinant avipox viruses for use in such vaccines.

It is a further object of this invention to provide a method forinducing an immunological response in avian and non-avian vertebrates toan antigen by inoculating the vertebrate with a synthetic recombinantavipox virus which, in the case of non-avian vertebrates such asmammals, cannot productively replicate in the animal with the productionof infectious virus. In this case, the virus is self-limiting, reducingthe possibility of spreading to non-vaccinated hosts.

It is a still further object of the invention to provide a method forinducing an immunological response in a vertebrate to an antigen, whichmethod comprises inoculating the vertebrate with a vaccine includingsynthetic recombinant avipox virus which comprises and expresses theantigenic determinant of a pathogen for said vertebrate.

It is another object of the invention to provide a method for expressinga gene product in a vertebrate by inoculating the vertebrate with arecombinant virus containing DNA which codes for and expresses the geneproduct without productive replication of the virus in the vertebrate.

It is yet another object of the invention to provide a method forinducing an immunological response in a vertebrate to an antigen byinoculating the vertebrate with a recombinant virus containing DNA whichcodes for and expresses the antigen without productive replication ofthe virus in the vertebrate.

STATEMENT OF THE INVENTION

In one aspect the present invention relates to a method for inducing animmunological response in a vertebrate to a pathogen by inoculating thevertebrate with a synthetic recombinant avipox virus modified by thepresence, in a nonessential region of the avipox genome, of DNA from anysource which codes for and expresses an antigen of the pathogen.

In a further aspect, the present invention is directed to a method forexpressing a gene product or inducing an immunological response to anantigen in a vertebrate with a recombinant virus which does notproductively replicate in the cells of the vertebrate but which doesexpress the gene product or the antigen in those cells.

In another aspect, the present invention is directed to syntheticrecombinant avipox virus modified by the insertion therein of DNA fromany source, and particularly from a non-avipox source, into anonessential region of the avipox genome. Synthetically modified avipoxvirus recombinants carrying exogenous (i.e. non-avipox) genes coding forand expressing an antigen, which recombinants elicit the production by avertebrate host of immunological responses to the antigen, and thereforeto the exogenous pathogen, are used according to the invention to createnovel vaccines which avoid the drawbacks of conventional vaccinesemploying killed or attenuated live organisms, particularly when used toinoculate non-avian vertebrates.

It must be noted again that avipox viruses can only productivelyreplicate in or be passaged through avian species or avian cell lines.The recombinant avipox viruses harvested from avian host cells, wheninoculated into a non-avian vertebrate such as a mammal in a manneranalogous to the inoculation of mammals by vaccinia virus, produce aninoculation lesion without productive replication of the avipox virus.Despite the failure of the avipox virus to productively replicate insuch an inoculated non-avian vertebrate, sufficient expression of thevirus occurs so that the inoculated animal responds immunologically tothe antigenic determinants of the recombinant avipox virus and also tothe antigenic determinants encoded in exogenous genes therein.

When used to inoculate avian species, such a synthetically recombinantavipox virus not only produces an immunological response to antigensencoded by exogenous DNA from any source which may be present therein,but also results in productive replication of the virus in the host withthe evocation of an expected immunological response to the avipox vectorper se.

Several investigators have proposed creating recombinant fowlpox,specifically viruses for use as veterinary vaccines for the protectionof fowl livestock. Boyle and Coupar, J. Gen. Virol. 67, 1591-1600(1986), and Binns et al., Isr. J. Vet. Med. 42, 124-127 (1986). Neitherproposals nor actual reports directed to the use of recombinant avipoxviruses as a method to induce specific immunity in mammals have beenuncovered.

Stickl and Mayr, Fortschr. Med. 97(40), pages 1781-1788 (1979) describethe injection of avipox, specifically fowlpox, virus into humans.However, these studies relate only to the use of ordinary fowlpox toenhance nonspecific immunity in patients suffering from the aftereffects of cancer chemotherapy. No recombinant DNA techniques areemployed. There is no teaching of an avipox into which DNA coding forantigens of vertebrate pathogens had been inserted, or of a method forinducing specific immunity in vertebrates. Instead, the prior artdepended upon a general and nonspecific tonic effect on the human host.

A more complete discussion of the basis of genetic recombination mayhelp in understanding how the modified recombinant viruses of thepresent invention are created.

Genetic recombination is in general the exchange of homologous sectionsof deoxyribonucleic acid (DNA) between two strands of DNA. (In certainviruses ribonucleic acid [RNA] may replace DNA). Homologous sections ofnucleic acid are sections of nucleic acid (RNA or DNA) which have thesame sequence of nucleotide bases.

Genetic recombination may take place naturally during the replication ormanufacture of new viral genomes within the infected host cell. Thus,genetic recombination between viral genes may occur during the viralreplication cycle that takes place in a host cell which is co-infectedwith two or more different viruses or other genetic constructs. Asection of DNA from a first genome is used interchangeably inconstructing the section of the genome of a second co-infecting virus inwhich the DNA is homologous with that of the first viral genome.

However, recombination can also take place between sections of DNA indifferent genomes that are not perfectly homologous. If one such sectionis from a first genome homologous with a section of another genomeexcept for the presence within the first section of, for example, agenetic marker or a gene coding for an antigenic determinant insertedinto a portion of the homologous DNA, recombination can still take placeand the products of that recombination are then detectable by thepresence of that genetic marker or gene.

Successful expression of the inserted DNA genetic sequence by themodified infectious virus requires two conditions.

First, the insertion must be into a nonessential region of the virus inorder that the modified virus remain viable. Neither fowlpox nor theother avipox viruses have as yet demonstrated nonessential regionsanalogous to those described for the vaccinia virus. Accordingly, forthe present invention nonessential regions of fowlpox were discovered bycleaving the fowlpox genome into fragments, then separating thefragments by size and inserting these fragments into plasmid constructsfor amplification. (Plasmids are small circular DNA molecules found asextra chromosomal elements in many bacteria including E. coli. Methodsfor inserting DNA sequences such as the genes for antigenic determinantsor other genetic markers into plasmids are well known to the art anddescribed in detail in Maniatis et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory New York [1982]). This wasfollowed by insertion of genetic markers and/or genes coding forantigens into the cloned fowlpox fragments. Those fragments whichdirected successful recombination, as proved by successful recovery ofthe genetic marker or antigens, were those which comprised DNA insertedinto a nonessential region of the fowlpox genome.

The second condition for expression of inserted DNA is the presence of apromoter in the proper relationship to the inserted DNA. The promotermust be placed so that it is located upstream from the DNA sequence tobe expressed. Because avipox viruses are not well characterized andavipox promoters have not previously been identified in the art, knownpromoters from other pox viruses are usefully inserted upstream of theDNA to be expressed as part of the present invention. Fowlpox promotersalso can be successfully used to carry out the methods and make theproducts of the invention. According to the present invention, fowlpoxpromoters, vaccinia promoters and entomopox promoters have been found topromote transcription in recombinant pox virus.

Boyle and Coupar, J. gen. Virol. 67, 1591, (1986) have publishedspeculation that vaccinia promoters "might be expected to operate in(fowlpox) virus." The authors located and cloned a fowlpox TK gene(Boyle et al., Virology 156, 355-365 [1987]) and inserted it into avaccinia virus. This TK gene was expressed, presumably because ofrecognition of the fowlpox TK promoter sequence by vaccinia polymerasefunctions. However, despite their speculation, the authors did notinsert any vaccinia promoter into a fowlpox virus nor observe anyexpression of a foreign DNA sequence present in a fowlpox genome. It wasnot known before the present invention that promoters from other poxviruses, such as vaccinia promoters, would in fact promote a gene in anavipox genome.

DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS

Fowlpox and canarypox viruses have been particularly used according tothe present invention as preferred avipox species to be modified byrecombination in incorporating exogenous DNA thereinto.

Fowlpox is a species of avipox which infects chickens in particular, butdoes not infect mammals. The fowlpox strain designated herein as FP-5 isa commercial fowlpox virus vaccine strain of chicken embryo originavailable from American Scientific Laboratories (Division of ScheringCorp.) Madison, Wis., United States Veterinary License No. 165, Ser. No.30321.

The fowlpox strain designated herein as FP-1 is a Duvette strainmodified to be used as a vaccine in one-day old chickens. The strain isa commercial fowlpox virus vaccine strain designated O DCEP 25/ CEP67/2309 October 1980 and is available from Institute Merieux, Inc.

Canarypox is another species of avipox. Analogously to fowlpox,canarypox particularly infects canaries, but does not infect mammals.The canarypox strain designated herein as CP is a commercial canarypoxvaccine strain designated LF2 CEP 524 24 10 75 and is available fromInstitute Merieux, Inc.

The DNA genetic sequences inserted into these avipox viruses by geneticrecombination according to the present invention include the Lac Z gene,of prokaryotic origin; the rabies glycoprotein (G) gene, an antigen of anon-avian (specifically mammalian) pathogen; the turkey influenzahemagglutinin gene, the antigen of a pathogenic avian virus other thanan avipox virus; the gp51,30 envelope gene of the bovine leukemia virus,a mammalian virus; the fusion protein gene of the Newcastle diseasevirus (Texas strain), an avian virus; the FeLV envelope gene of thefeline leukemia virus, a mammalian virus; the RAV-1 env gene of the rousassociated virus which is an avian virus/poultry disease; thenucleoprotein (NP) gene of the Chicken/Pennsylvania/1/83 influenzavirus, an avian virus; the matrix gene and peplomer gene of theinfectious bronchitis virus (strain Mass 41), an avian virus; and theglycoprotein D gene (gD) of herpes simplex virus; a mammalian virus.

Isolation of the Lac Z gene is described by Casadaban et al., Methods inEnzymology 100, 293-308 (1983). The structure of the rabies G gene isdisclosed, for example, by Anilionis et al., Nature 294, 275-278 (1981).

Its incorporation into vaccinia and expression in this vector arediscussed by Kieny et al., Nature 312, 163-166 (1984). The turkeyinfluenza hemagglutinin gene is described by Kawaoka et al., Virology158, 218-227 (1987). The bovine leukemia virus gp51,30 env gene has beendescribed by Rice et al., Virology 138, 82-93 (1984). The fusion gene ofthe Newcastle disease virus (Texas strain) is available from InstituteMerieux, Inc., as plasmid pNDV 108. The feline leukemia virus env genehas been described by Guilhot et al., Virology 161, 252-258 (1987). Therous associated virus type 1 is available from Institute Merieux, Inc.,as two clones, penVRVIPT and mp19env (190). Chicken influenza NP gene isavailable from Yoshihiro Kawaoka of St. Jude Children's ResearchHospital as plasmid pNP 33. An infectious bronchitis virus cDNA clone ofthe IBV Mass 41 matrix gene and peplomer gene are available fromInstitute Merieux, Inc. as plasmid pIBVM63. The herpes simplex virus gDgene is described in Watson et al., Science 218, 381-384 (1982).

The recombinant avipox viruses described in more detail belowincorporate one of three vaccinia promoters. The Pi promoter, from theAva I H region of vaccinia, is described in Wachsman et al., J. of Inf.Dis. 155, 1188-1197 (1987). More in particular, this promoter is derivedfrom the Ava I H(Xho I G) fragment of the L-variant WR vaccinia strain,in which the promoter directs transcription from right to left. The maplocation of the promoter is approximately 1.3 Kbp (kilobase pair) fromthe left end of Ava IH, approximately 12.5 Kbp from the left end of thevaccinia genome, and about 8.5 Kbp left of the Hind III C/N junction.The sequence of the promoter is:

(GGATCCC)-ACTGTAAAAATAGAAACTATAATCATATAATAGTGTAGGTTGGTAGTAGGGTACTCGTGATTAATTTTATTGTTAAACTTG-(AATTC),

wherein the symbols in parentheses are linker sequences.

The Hind III H promoter (also "HH" and "H6" herein) was defined bystandard transcriptional mapping techniques. It has the sequence

    __________________________________________________________________________    ATTCTTTATTCTATACTTAAAAAATGAAAA                                                TAAATACAAAGGTTCTTGAGGGTTGTGTTAAATTGAAAGCGAGAAATAATCATAAATT                    ATTTCATTATCGCGATATCCGT                                                        TAAGTTTGTATCGTAATG.                                                           __________________________________________________________________________

The sequence is identical with that described as being upstream of openreading frame H6 by Rosel et al., J. Virol. 60, 436-449 (1986).

The 11K promoter is as described by Wittek, J. Virol. 49, 371-378 (1984)and Bertholet, C. et al., Proc. Natl. Acad. Sci. USA 82, 2096-2100(1985).

The recombinant avipox viruses of the present invention are constructedin two steps known in the art and analogous to those disclosed inaforementioned U.S. Pat. No. 4,603,112 for creating syntheticrecombinants of the vaccinia virus.

First, the DNA gene sequence to be inserted into the virus is placedinto an E. coli plasmid construct into which DNA homologous to a sectionof nonessential DNA of the avipox virus has been inserted. Separately,the DNA gene sequence to be inserted is ligated to a promoter. Thepromoter-gene linkage is then inserted into the plasmid construct sothat the promoter-gene linkage is flanked on both ends by DNA homologousto a nonessential region of avipox DNA. The resulting plasmid constructis then amplified by growth within E. coli bacteria. (Plasmid DNA isused to carry and amplify exogenous genetic material, and this method iswell known in the art. For example, these plasmid techniques aredescribed by Clewell, J. Bacteriol. 110, 667-676 (1972). The techniquesof isolating the amplified plasmid from the E. coli host are also wellknown in the art and are described, for instance, by Clewell et al. inProc. Natl. Acad. Sci. U.S.A. 62, 1159-1166 (1969).)

The amplified plasmid material isolated after growth within E. coli isthen used for the second step. Namely, the plasmid containing the DNAgene sequence to be inserted is transfected into a cell culture, e.g.chick embryo fibroblasts, along with the avipox virus (such as fowlpoxstrain FP-1 or FP-5). Recombination between homologous fowlpox DNA inthe plasmid and the viral genome respectively gives an avipox virusmodified by the presence, in a nonessential region of its genome, ofnon-fowlpox DNA sequences.

A better understanding of the present invention and of its manyadvantages will be had from the following examples, given by way ofillustration.

EXAMPLE 1 TRANSIENT EXPRESSION ASSAYS DEMONSTRATING RECOGNITION OFVACCINIA PROMOTERS BY FOWLPOX RNA TRANSCRIPTION FACTORS

A number of plasmid constructions were made containing the Hepatitis Bvirus surface antigen (HBSAg) coding sequence linked to vaccinia viruspromoter sequences. Fifty ug of each plasmid were transfected onto CEFcells infected with 10 pfu per cell of fowlpox virus or vaccinia virus.Infection was allowed to proceed for 24 hours and cells were then lysedby three successive cycles of freezing and thawing.

The amount of HBSAg in the lysate was estimated using the commerciallyavailable AUSRIA II-¹²⁵ I kit from Abbott Laboratories, DiagnosticDivision. The presence or absence of HBSAg is expressed as a ratio ofthe net counts (sample minus background) of the unknown to a negativecutoff value pre- determined by the manufacturer. This results in a P/N(positive/negative) ratio. The results are shown in Table I.

Three different vaccinia promoter sequences were used: the Pi promoter,recognized early in vaccinia infection before DNA replication; the 11Kpromoter, recognized late in vaccinia infection after the onset of DNAreplication; and the Hind III H (HH) promoter, recognized both early andlate in vaccinia infection. These promoters are described earlierherein.

The data indicate that HBSAg produced in the lysates of infected cellsis the result of recognition of vaccinia promoters by either fowlpox orvaccinia transcriptional factors.

                  TABLE I                                                         ______________________________________                                        Plasmid     Virus     Description                                                                              P/N Ratio                                    ______________________________________                                        pMP 131piR.sub.2                                                                          Fowlpox   SAg linked to                                                                            1.8                                                      Vaccinia  Pi promoter                                                                              9.1                                          pMPK 22.13S Fowlpox   SAg linked to                                                                            14                                                       Vaccinia  11K promoter                                                                             2                                            pPDK 22.5   Fowlpox   SAg linked to                                                                            92.6                                                     Vaccinia  11K promoter                                                                             5.6                                          pRW 668     Fowlpox   SAg linked to                                                                            77                                                       Vaccinia  HH promoter                                                                              51.4                                         (no plasmid)                                                                              Fowlpox              1.1                                          (no plasmid)                                                                              Vaccinia             1.3                                          pMPK 22.13S (no virus)           1.3                                          ______________________________________                                    

EXAMPLE 2 CONSTRUCTION OF RECOMBINANT FOWLPOX VIRUS vFP-1 CONTAINING THELAC Z GENE

A fragment in a nonessential region of the fowlpox virus was located andisolated as follows.

The nuclease Bal 31 was employed to remove the single stranded terminalhairpin loops of FP-5 DNA. The Klenow (large) fragment of DNA polymeraseI was used to create blunt ends. Following removal of the loops, thefragments were generated by restriction endonuclease digestion with BglII. This digestion produced a series of FP-5 fragments which wereseparated by agarose gel electrophoresis.

An 8.8 Kbp Bgl II blunt ended fragment was isolated and ligated into acommercially available plasmid, pUC 9, which had been cleaved with BamHI and Sma I. The resulting plasmid was designated pRW 698.

To decrease the size of the fowlpox fragment, this plasmid was cleavedwith Hind III to create two further fragments. A 6.7 Kbp fragment wasdiscarded and the remaining 4.7 Kbp fragment was ligated onto itself toform a new plasmid designated pRW 699.

To incorporate an 11K promoted Lac Z gene into this plasmid, pRW 699 wascut with EcoRV, which cleaves the plasmid at only one site. The 11Kpromoted Lac Z segment was then inserted as a blunt ended PstI-Bam HIfragment, creating a new plasmid designated pRW 702. The Lac Z clone isfrom pMC 1871, as described in Casadaban et al., loc. cit. The 11Kpromoter was ligated to the eighth codon of the Lac Z gene via a Bam HIlinker.

With recombination techniques like those taught for vaccinia in U.S.Pat. No. 4,603,112, the pRW 702 plasmid was then recombined with thefowlpox virus FP-5 growing on chick embryo fibroblasts (CEF) using thefollowing procedures to generate vFP-1. Fifty ug of pRW 702 DNA wasmixed in a final volume of 100 ul with 0.5 ug of whole genome fowlpoxDNA. To this were added 10 ul of 2.5 M CaCl₂ and 110 ul of 2 x HEBSbuffer (pH 7) prepared from:

40 mM Hepes

300 mM NaCl

1.4 mM Na₂ HPO₄

10 mM KCl

12 mM dextrose.

After 30 minutes at room temperature, 200 ul of a fowlpox virus pooldiluted to give 5 pfu/cell were added and the mixture inoculated onto 60mm dishes containing a primary CEF monolayer. 0.7 ml of Eagles mediumcontaining 2% fetal bovine serum (FBS) was also added at this time. Theplates were incubated at 37° C. for 2 hours, after which an additional 3ml of Eagles medium containing 2% FBS was added and the plates incubatedfor 3 days. Cells were lysed by three successive cycles of freezing andthawing and progeny virus was then assayed for the presence ofrecombinants.

Proof of successful insertion by recombination of the 11K-promoted Lac Zgene into the genome of fowlpox FP-5 was obtained by testing forexpression of the Lac Z gene. The Lac Z gene codes for the enzymeBeta-galactosidase, which cleaves the chromogenic substrate5-bromo-4-chloro-3-indolyl-Beta-D-galactoside (X-gal) releasing a blueindolyl derivative. Blue plaques were selected as positive recombinants.

The successful insertion of Lac Z into the genome of fowlpox FP-5 andits expression were also confirmed by immune precipitation of theBeta-galactosidase protein with commercially available antisera andstandard techniques using vFP-1 infected CEF, BSC (monkey kidney cellline--ATCC CCL26), VERO (monkey kidney cell line--ATCC CCL81), and MRC-5(human diploid lung cell line--ATCC CCL171).

The expression of Beta-galactosidase by the recombinant virus vFP-1 wasfurther confirmed in vivo by inoculating rabbits and mice with the virusand successfully measuring a post-inoculation rise in the titers ofantibodies directed against the Beta-galactosidase protein in the serumof the inoculated animals.

In particular, the recombinant vFP-I was purified from host cellcontaminants and inoculated intradermally at two sites on each side oftwo rabbits. Each rabbit received a total of 10⁸ pfu.

Animals were bled at weekly intervals and the sera used in an ELISAassay using a commercially available preparation of purifiedBeta-galactosidase as an antigen source.

Both rabbits and mice inoculated with the recombinant vFP-1 produced animmune response to the Beta-galactosidase protein as detected in anELISA assay. In both species the response was detectable by one weekpost-inoculation.

EXAMPLE 3 CONSTRUCTION FROM FOWLPOX VIRUS FP-5 OF THE RECOMBINANT VIRUSvFP-2 CONTAINING THE RABIES G GENE AND LAC Z

A 0.9 Kbp Pvu II fragment was obtained from FP-5 and inserted bystandard techniques between the two Pvu II sites in pUC 9. The resultingconstruct, designated pRW 688.2, has two Hinc II sites, approximately 30bp apart, asymmetric within the Pvu II fragment and thus forming a longarm and a short arm of the fragment.

Oligonucleotide adapters were inserted between these Hinc II sites usingknown techniques to introduce Pst I and Bam HI sites, thus creatingplasmid pRW 694.

This plasmid was now cleaved with Pst I and Bam HI and the Lac Z genehaving a linked 11K vaccinia promoter described earlier was inserted tocreate the new plasmid pRW 700.

To create a Pi-promoted rabies G gene, the Bgl II site which is5'-proximal to the rabies gene (cf. Kieny et al., loc. cit.) was bluntended and ligated to the filled-in Eco RI site of the Pi promoterdescribed earlier.

This construct was inserted at the Pst I site of pRW 700 to createplasmid pRW 735.1 having therein the foreign gene sequence Pi-rabiesG-11K-Lac Z. This insert is so oriented within the plasmid that the longPvu II-Hinc II arm of the FP-5 donor sequence is 3' to the Lac Z gene.

The resulting final construct was recombined with fowlpox virus FP-5 byinfection/transfection of chick embryo fibroblasts by the methodspreviously described to create recombinant fowlpox virus vFP-2. Thisrecombinant virus was selected by X-gal staining.

The proper insertion and expression of both the Lac Z marker gene andthe rabies G gene were verified by a number of additional methodsdescribed below.

Immunofluorescent localization of the rabies antigen by specificantibodies successfully demonstrated rabies antigen expression on thesurface of avian and non-avian cells infected with vFP-2 virus.

As earlier, the expression of rabies antigen and Beta-galactosidase byavian and non-avian cells infected with the vFP-2 virus was confirmed bythe immune precipitation method.

Further proof that the vFP-2 embodiment of this invention is asuccessful recombinant virus carrying the genes for rabies G andBeta-galactosidase was obtained by inoculating two rabbits with vFP-2virus. Both rabbits were inoculated intradermally with 1×10⁸ pfu perrabbit of vFP-2. Both of these rabbits produced typical pox lesions. Therabbits were bled at weekly intervals and sera were tested by ELISA todetect the presence of antibody specific for the rabies glycoprotein andthe Beta-galactosidase protein.

As reported in Table II below, rabbit 205 showed detectable levels ofanti-Beta-galactosidase antibody by the ELISA test at one weekpost-inoculation. This rose at two weeks to a titer of 1 in 4000 whichwas maintained to five weeks post inoculation. Using the antigen captureELISA assay, sera from rabbit 205 showed detectable levels ofanti-rabies antibodies from 3 to 10 weeks post-inoculation.

                  TABLE II                                                        ______________________________________                                        Antibody Production by Rabbit 205 Against Rabies                              Antigen and Beta-Galactosidase Protein                                                              Antibody Titer                                                                (Reciprocal of Serum                                    Time                  Dilution)                                               ______________________________________                                        Prebleed   anti-B-galactosidase                                                                          0                                                  Week 1                    500                                                 Weeks 2-5 (each)          4000                                                Week 6                    500                                                 Week 9                    250                                                 Prebleed   anti-rabies     0                                                  Week 3                    200                                                 Week 6                    200                                                 Week 10                   100                                                 ______________________________________                                    

EXAMPLE 4A CONSTRUCTION FROM FOWLPOX VIRUS FP-1 OF RECOMBINANT VIRUSvFP-3 CONTAINING PROMOTED RABIES G GENE

This embodiment demonstrates that the rabies G gene is fully expressedby fowlpox strains other than FP-5, specifically by another strain offowlpox virus designated FP-1.

As in Example 3, a 0.9 Kbp Pvu II fragment was obtained from FP-1 on theassumption that, as in FP-5, the fragment would contain a nonessentialregion.

This fragment was inserted between the two Pvu II sites of pUC 9,generating a plasmid designated pRW 731.15R.

This plasmid has two Hinc II sites, approximately 30 bp apart,asymmetric within the Pvu II fragment and thus forming a long arm and ashort arm of the fragment.

A commercially available Pst linker

(5')-CCTGCAGG-(3')

was inserted between the two Hinc II sites creating plasmid pRW 7.41.

An HH-promoted rabies G gene was inserted into this plasmid at the Pst Isite, to generate the new plasmid pRW 742B. By recombination of thisplasmid with FP-1 by infection/transfection of CEF cells, virus vFP-3was obtained.

The ATG translational initiation codon of the open reading framepromoted by the HH promoter was superimposed on the initiation codon ofthe rabies G gene using a synthetic oligonucleotide spanning the EcoRVsite in the HH promoter and the Hind III site in the rabies G gene.

The 5' end of this HH-promoted rabies gene was modified by knowntechniques to contain a Pst I site and the construct was then ligatedinto the Pst I site of pRW 741 to create pRW 742B. The orientation ofthe construct in the plasmid is the same as in pRW 735.1 discussedearlier in Example 3.

Recombination was carried out as described in Example 2. The resultingrecombinant is called vFP-3.

The expression of rabies antigen by both avian and non-avian cellsinfected with the vFP-3 virus was confirmed by the immune precipitationand immunofluorescence techniques earlier described.

Further proof that the vFP-3 embodiment of this invention is asuccessful recombinant virus expressing the genes for rabies G wasobtained by intradermally inoculating pairs of rabbits with therecombinant virus. Two rabbits were inoculated intradermally with 1×10⁸pfu of vFP-3 per rabbit. Both of these rabbits produced typical poxlesions reaching maximum size 5-6 days post-inoculation. The rabbitswere bled at weekly intervals and sera were tested by ELISA to detectthe presence of antibody specific for the rabies glycoprotein.

Each of five rats was also inoculated intradermally with 5×10⁷ pfu ofvFP-3. Lesions resulted in all animals.

Both rabbits and rats produced detectable levels of antibody specific torabies by two weeks after inoculation. Two control rabbits inoculatedintradermally with the parental FP-1 virus had no detectable levels ofanti-rabies antibody.

To exclude the possibility that the antibody response was due to theintroduction of rabies antigen adventitiously carried with the inoculumvirus or integrated into the membrane of the recombinant fowlpox virus,rather than being caused by de novo synthesis of the rabies antigen bythe recombinant virus in the animal as proposed, the vFP-3 virus waschemically inactivated and inoculated into rabbits.

The purified virus was inactivated overnight at 4° C. in the presence of0.001% of beta propiolactone and then pelleted by centrifugation. Thepelleted virus was collected in 10 nM Tris buffered saline, sonicated,and titrated to assure that no infectious virus remained. Two rabbitswere inoculated intradermally with inactivated vFP-3 and two with anequivalent amount of untreated recombinant. Lesion sizes were monitored.

Both rabbits receiving untreated vFP-3 developed typical pox lesionsgraded as 4-5+ at 5 days post-inoculation. Rabbits inoculated withinactivated virus also developed lesions, but these were graded as 2+ at5 days post-inoculation.

Rabbits were bled at weekly intervals and the sera tested by ELISA forthe presence of rabies specific antibody and fowlpox specificantibodies. The results are shown in Table III below.

                  TABLE III                                                       ______________________________________                                        Live vFP-3                                                                    Rabbit:                                                                              No. 295   No. 318   Inactivated vFP-3                                  Antibody                                                                             Ra-           Ra-       No. 303  No. 320                               Tested:                                                                              bies   FP     bies FP   Rabies                                                                              FP   Rabies                                                                              FP                            ______________________________________                                        Week P.I                                                                      0        0      0      0    0  0       0  0       0                           2       250   4000    500 4000 0      50  0     1000                          3      1000   4000    500 4000 0     4000 0     2000                          4      1000   4000   2000 4000 0     4000 0     2000                          5      4000   4000   2000 4000 0     2000 0     4000                          6      4000   4000   4000 4000 0     2000 0     2000                          ______________________________________                                    

In this test the titer end point (expressed as the reciprocal of theserum dilution) was arbitrarily set at 0.2 after the absorbance valuesof all pre-challenge sera were subtracted. Both rabbits 295 and 318receiving the live virus developed an immune response to the rabiesglycoprotein and to fowlpox virus antigens. Rabbits 303 and 320 alsodeveloped an immune response to fowlpox virus antigens although thetiter was lower. Neither of these rabbits developed a detectableresponse to the rabies glycoprotein.

This finding signifies that the immune response produced in the rabbitis due to the de novo expression of the rabies glycoprotein gene carriedin the recombinant virus and is not a response to any adventitiousglycoprotein carried in the inoculum virus.

EXAMPLE 4B CONSTRUCTION FROM FOWLPOX VIRUS FP-1 OF THE RECOMBINANT VIRUSvFP-5 CONTAINING UNPROMOTED RABIES G GENE

Expression of a foreign gene inserted by recombination into the fowlpoxgenome requires the presence of a promoter. This was demonstrated by thecreation of a further recombinant, vFP-5, identical to vFP-3 except forthe omission of the HH promoter. The presence of the rabies gene in thisrecombinant was confirmed by nucleic acid hybridization. However, norabies antigen was detected in CEF cell cultures infected by the virus.

EXAMPLE 5 IN VITRO PASSAGING EXPERIMENTS TO DETERMINE WHETHER FOWLPOXVIRUS REPLICATES IN NON-AVIAN CELLS

An experiment was performed in which three cell systems, one avian andtwo non-avian, were inoculated with the parental FP-1 strain or therecombinant vFP-3. Two dishes each of CEF, MRC-5, and VERO,respectively, were inoculated with FP-1 or vFP-3 at an inputmultiplicity of 10 pfu per cell.

At three days, one dish each was harvested. The virus was released bythree successive cycles of freezing and thawing and re-inoculated onto afresh monolayer of the same cell line. This was repeated for sixsequential passages and, at the end of the experiment, samples of eachpassage were titrated for virus infectivity on CEF monolayers.

The results are shown in Table IVA and indicate that the serial passageof both FP-1 and vFP-3 is possible in CEF cells but not in either of thetwo non-avian cells lines. Infectious virus is not detectable after 3 or4 passages in VERO or MRC-5 cells.

The second dish was used to determine if virus, not detectable by directtitration, could be detected after amplification in the permissive CEFcells. At three days, cells on the second dish were harvested byscraping and a third of the cells lysed and inoculated onto a fresh CEFmonolayer. When full cytopathic effect (CPE) was reached or at 7 dayspost-infection, the cells were lysed and the virus yield titrated. Theresults are shown in Table IVB. When passage in CEF cells was used toamplify any virus present, the virus could not be detected after four orfive passages.

Attempts to establish persistently infected cells failed.

In a further attempt to detect evidence of continued viral expression innon-avian cells, the samples used for viral titration above were used ina standard immunodot assay in which anti-fowlpox antibody andanti-rabies antibody were used to detect the presence of the respectiveantigens. The results of these assays confirm the titration results.

                  TABLE IVA                                                       ______________________________________                                        Passaging Experiment                                                          Inoculum Virus   FP-1                vFP-3                                    Cell Type CEF    VERO    MRC-5 CEF   VERO  MRC-5                              ______________________________________                                        Pass 1    6.6.sup.a                                                                            4.8     4.9   6.6   5.4   6.2                                2         6.7    2.9     3.7   6.5   4.2   5.1                                3         6.4    1.4     1.0   6.4   1.7   4.4                                4         6.1    N.D.sup.b                                                                             N.D   6.2   N.D   1.0                                5         6.4    N.D     N.D   6.3   N.D   N.D                                6         5.7    N.D     N.D   5.9   N.D   N.D                                ______________________________________                                         .sup.a titer of virus expressed as log.sub.10 pfu per ml.                     .sup.b not detectable.                                                   

                  TABLE IVB                                                       ______________________________________                                        Amplification Experiment                                                      Inoculum Virus   FP-1                vFP-3                                    Cell Type CEF    VERO    MRC-5 CEF   VERO  MRC-5                              ______________________________________                                        Pass 1    6.4.sup.a                                                                            6.2     6.4   6.5   6.3   6.4                                2         7.5    6.3     6.0   6.5   6.3   5.5                                3         6.2    6.7     5.3   5.9   6.1   6.3                                4         5.6    4.6     3.9   5.7   4.8   5.8                                5         6.3    4.1     N.D   6.1   4.7   4.7                                6         6.2    N.D.sup.b                                                                             N.D   6.2   N.D   N.D                                ______________________________________                                         .sup.a titer of virus expressed as log.sub.10 pfu per ml.                     .sup.b not detectable.                                                   

EXAMPLE 6 ADDITIONAL RECOMBINANTS OF FOWLPOX FP-1: vFP-6, vFP-7, vFP-8,AND vFP-9

Recombinant viruses vFP-6 and vFP-7 were constructed by the followingprocedure.

A 5.5 Kbp Pvu II fragment of FP-1 was inserted between the two Pvu IIsites in pUC 9 to create the plasmid pRW 731.13. This plasmid was thencut at a unique Hinc II site and blunt ended HH-promoted rabies G geneinserted to create plasmids pRW 748A and B, representing oppositeorientations of the insert. Plasmids pRW 748A and B were then usedseparately to transfect CEF cells along with FP-1 virus to produce vFP-6and vFP-7, respectively, by recombination. This locus is now designatedas locus f7.

A 10 Kbp Pvu-II fragment of FP-1 was inserted between the two Pvu IIsites of pUC 9 to create pRW 731.15. This plasmid was then cut at aunique Bam HI site and then an 11K promoted Lac Z gene fragment wasinserted, generating pRW 749A and B, representing opposite orientationsof the insert. Recombination of these donor plasmids with FP-1 resultedin vFP-8 and vFP-9, respectively. This locus is now designated as locusf8.

vFP-8 and vFP-9 expressed the Lac Z gene as detected by X-gal. vFP-6 andvFP-7 expressed the rabies G gene as detected by rabies-specificantiserum.

EXAMPLE 7 IMMUNIZATION WITH vFP-3 TO PROTECT ANIMALS AGAINST CHALLENGEWITH LIVE RABIES VIRUS

Groups of 20 female SPF mice, 4-6 weeks, were inoculated with 50 ul ofvFP-3 in the footpad in doses ranging from 0.7 to 6.7 TCID₅₀ per mouse.(The TCID₅₀ or tissue culture infectious dose is that dose at which 50percent of tissue culture cells suffer cytopathic effect). At 14 dayspost-vaccination 10 mice in each group were sacrificed and serum samplescollected for assay in the RFFI test. The remaining 10 mice werechallenged by inoculation of 10 LD₅₀ of CVS strain rabies by theintracerebral route and survivors calculated at 14 days post-challenge.

The results are shown in Table VA below.

                  TABLE VA                                                        ______________________________________                                                      Rabies Antibody                                                 Dose vFP-3    Titer Log.sub.10                                                Log.sub.10 TCID.sub.50                                                                      Dilution*    Survival                                           ______________________________________                                        6.7           1.9          8/10                                               4.7           1.8          0/10                                               2.7           0.4          0/10                                               0.7           0.4          0/10                                               ______________________________________                                         *As measured in the RFFI (Rapid Fluorescent Focus Inhibition) test,           Laboratory Techniques in Rabies, Third Ed., 354-357, WHO Geneva.         

The experiment was repeated with 12.5 LD₅₀ of challenge rabies virus.The results are shown in Table VB below.

                  TABLE VB                                                        ______________________________________                                                      Rabies Antibody                                                 Dose vFP-3    Titer Log.sub.10                                                Log.sub.10 TCID.sub.50                                                                      Dilution*    Survival                                           ______________________________________                                        6.7           2.8           5/10                                              4.7           2.1           2/10                                              2.7           0.6          0/8                                                0.7           0.6          0/8                                                ______________________________________                                    

Two dogs and two cats were immunized with a single subcutaneousinoculation of 8 log₁₀ TCID₅₀ of the recombinant vFP-3. In addition, twodogs and four cats of equivalent age and weight were held asnon-vaccinated controls. All animals were bled at weekly intervals. Atday 94 each dog was challenged by inoculation in the temporal musclewith two doses of 0.5 ml of a salivary gland homogenate of the NY strainof rabies virus available from Institut Merieux, Inc. The total dosecorresponded to 10000 mouse LD₅₀ by an intracerebral route. The six catswere similarly challenged by inoculation in the neck muscle with twodoses of 0.5 ml of the same virus suspension. The total dose per animalcorresponded to 40000 mouse LD₅₀ by an intracerebral route. The animalswere observed daily. All non-vaccinated animals died on the dayindicated in Table VI with rabies symptoms. The vaccinated animalssurvived challenge and were observed for three weeks after the death ofthe last control animal. The results are shown in Table VI below.

                  TABLE VI                                                        ______________________________________                                                    Titer at Days                                                                 post-Inoculation                                                                           Survival/                                            Animal  Vaccination                                                                             0     14   21  28  94  Time of Death                        ______________________________________                                        Cat  7015   vFP-3.sup.a                                                                             0   2.2.sup.b                                                                          2.4 2.4 1.5 +                                       7016   vFP-3     0   1.7  1.9 2.0 1.3 +                                       8271   c.sup.c   0   0    0   0   0   .sup. d/13.sup.d                        T10    c         0   0    0   0   0   d/12                                    T41    c         0   0    0   0   0   d/13                                    T42    c         0   0    0   0   0   d/12                               Dog  426    vFP-3     0   0.8  1.0 1.1 1.2 +                                       427    vFP-3     0   1.5  2.3 2.2 1.9 +                                       55     c         0   0    0   0   0   d/15                                    8240   c         0   0    0   0   0   d/16                               ______________________________________                                         .sup.a Both cats and dogs vaccinated with vFP3 received 8 log.sub.10          TCID.sub.50 by the subcutaneous route.                                        .sup.b Titer expressed as log.sub.10 highest serum dilution giving greate     than 50% reduction in the number of fluorescing wells in an RFFI test.        .sup.c Non-vaccinated control animals.                                        .sup.d Animal died/day of death postchallenge.                           

In further experiments, the recombinant viruses vFP-2 and vFP-3 wereinoculated into cattle by several different routes.

Inoculated animals were tested for anti-rabies antibody at days 6, 14,21, 28, and 35. As shown in following Table VIIA, all animals showed aserological response to the rabies antigen.

                  TABLE VIIA                                                      ______________________________________                                        Antibody Titers in Mammals Inoculated with vFP-3                              Anti-rabies Neutralizing Antibodies                                           RFFI Test Log.sub.10 Dilut.                                                             Day                                                                 Cattle No.  0       6      14   21    28   35                                 ______________________________________                                        7.3 log.sub.10 TCID.sub.50                                                                NEG     0.6    2    1.7   1.8  1.7                                1420 (intraderm)                                                              8 log.sub.10 TCID.sub.50                                                                  NEG     1.6    2.2  2.1   2.1  1.9                                1419 (subcut)                                                                 8 log.sub.10 TCID.sub.50                                                                  NEG     0.9    2.2  2.2   1.8  1.7                                1421 (intramusc)                                                              7.3 log.sub.10 TCID.sub.50                                                                NEG     0.9    1.1+ 1+    1+   1.1+                               1423 (intramusc)                                                              ______________________________________                                         +Not significant                                                         

All the cattle were revaccinated with 8 log₁₀ TCID₅₀ at day 55post-inoculation and exhibited an anamnestic response to the rabiesantigen. In the booster revaccination, all cattle were inoculatedsubcutaneously except No. 1421, which was again inoculatedintramuscularly. RFFI titers were determined on days 55, 57, 63, 70, 77,and 86. The results are shown in Table VIIB.

                  TABLE VIIB                                                      ______________________________________                                                 Day                                                                  Cattle No. 55    57        63  70     77  86                                  ______________________________________                                        1419       1.7   1.5       2.9 2.9    2.6 2.9                                 1420       1.0   0.5       1.9 2.3    2.2 2.0                                 1421       1.3   1.2       2.9 2.7    2.5 2.5                                 1423       1.0   0.7       2.4 2.5    2.5 2.2                                 ______________________________________                                    

All data are for vFP-3 except for animal 1423, which is for vFP-2.

Cattle, cats, and rabbits were also inoculated intradermally with knownamounts of fowlpox virus and scabs were collected from the animals afterabout a week. These were ground, suspended in saline, and titrated todetermine virus levels.

Only residual amounts of infectious virus could be recovered. Thisdemonstrates that no productive infection occurred in vivo.

EXAMPLE 8 INOCULATION OF CHICKENS WITH vFP-3

The recombinant fowlpox virus vFP-3 was inoculated into chickens todemonstrate the expression of foreign DNA by a recombinant fowlpox virusin a system permitting productive replication of the vector.

White leghorn chickens were inoculated intramuscularly with 9 log₁₀TCID₅₀ vFP-3 or 3 log₁₀ TCID₅₀ vFP-3 by wing transfixion. Blood sampleswere taken for an RFFI test for rabies antibody titer 21 days aftervaccination. Day 21 titers in inoculated chickens were significantlyhigher than day 21 titers in controls. Namely, the average titer in theuninfected controls was 0.6; the average in the intramuscularlyinoculated birds was 1.9; that in the transfixed birds was 1.2.

EXAMPLE 9 RECOMBINANT FOWLPOX vFP-11 EXPRESSING TURKEY INFLUENZA H5 HAANTIGEN

Avian species can be immunized against avian pathogens using therecombinant avipox viruses of the invention.

Thus, novel plasmid pRW 759 (described below), derived from fowlpoxvirus FP-1 and containing the Hind III H-promoted hemagglutinin gene(H5) of A/turkey/Ireland/1378/83 (TYHA), was used to transfect CEF cellsconcurrently infected with parent virus FP-1. Recombinant fowlpox virusvFP-11 was obtained by the techniques described earlier herein.

The synthesis of a hemagglutinin molecule by VFP-11 infected cells wasconfirmed by immune precipitation from metabolically radiolabeledinfected cell lysates using specific anti H5-antibody and standardtechniques. The specific immune precipitation of precursor hemagglutininwith a molecular weight of approximately 63 kd (kilodaltons) and twocleavage products with molecular weights of 44 and 23 kd wasdemonstrated. No such proteins were precipitated from a lysate ofuninfected CEF cells or parental virus, FP-1, infected cells.

To determine that the HA molecule produced in cells infected with therecombinant fowlpox, vFP-11, was expressed on the cell surface,immunofluorescence studies were performed. CEF cells infected with therecombinant fowlpox virus, vFP-11, showed strong surface fluorescentstaining. In cells infected with the parental virus, FP-1, nofluorescence was detected.

Plasmid pRW 759 was created as follows:

pRW 742B (cf. Example 4) is linearized by partial digestion with Pst Iand the fragment is recut with EcoRV to remove the rabies G gene,leaving the HH promoter on the remaining fragment of about 3.4 Kbp. Thisis treated with alkaline phosphatase and a synthetic oligonucleotide wasinserted for joining of the HH promoter with TYHA at ATG to generate pRW744.

This plasmid was linearized by partial digestion with Dra I, the linearfragment was cut with Sal I, and the larger fragment was reisolated andtreated with alkaline phosphatase.

Finally, pRW 759 was generated by inserting into the pRW 744 vector theisolated Sal I-Dra I coding sequence of TYHA, disclosed by Kawaoka etal., Virology 158, 218-227 (1987).

EXAMPLE 10 IMMUNIZATION WITH vFP-11 TO PROTECT BIRDS AGAINST CHALLENGEWITH LIVE INFLUENZA VIRUS

In order to assess the immunogenicity of the recombinant fowlpox virusvFP-11, vaccination and challenge experiments were performed in chickensand turkeys.

Specific pathogen free white leghorn chickens were vaccinated at 2 daysand 5 weeks of age by wing web puncture with a double needle used forcommercial vaccination of poultry with fowlpox virus. Approximately 2 ulcontaining 6×10⁵ pfu of vPF-11 was given to each bird. The older birdswere bled before vaccination, and all birds were bled prior to challengeand two weeks later.

For comparative purposes, a second group of chickens was vaccinated witha conventional H5 vaccine consisting of an inactivated H5N2 strain in awater-in-oil emulsion.

Inactivated H5N2 vaccine was prepared from A/Mallard/NY/189/82 (H5N2)influenza virus grown in 11 day embryonated chicken eggs; the infectedallantoic fluid with an HA titer of 800/0.1 ml and infectivity titer of10⁸.5 /0.1 ml was inactivated with 0.1% propiolactone and suspended inwater-in-oil emulsion as described in Stone et al., Avian Dis. 22,666-674 (1978) and Brugh et al., Proc. Second Inter. Sym. on AvianInfluenza, 283-292 (1986). The vaccine in 0.2 ml volume was administeredto 2 day and 5 week old SPF white leghorn chickens by the subcutaneousroute, under the skin on the inside of the thigh muscle.

A third and fourth group of chickens received parental virus FP-1 or novaccine, respectively.

Chickens were challenged with approximately 10³ LD₅₀ of the highlypathogenic A/Turkey/Ireland/1378/83 (H5N8) or A/Chick/Penn/1370/83(H5N2) influenza virus by administering 0.1 ml to the nares of eachbird. Two day old birds were challenged 6 weeks after vaccination and 5week old birds were challenged at 5 weeks post vaccination. The birdswere observed daily for disease signs indicated by swelling and cyanosisof the face and comb and hemorrhage of the legs (such birds couldfrequently not stand), paralysis and death. Most deaths occurred between4 and 7 days after infection. Tracheal and cloacal swabs were taken ofeach live chicken 3 days after infection and screened for virus byinoculation into embryonated eggs. The chickens inoculated with eitherwildtype or recombinant fowlpox virus developed typical lesions on thewing web. Pustules formed by the third day at the site of each needlestick, cellular infiltration followed with scab formation and recoveryby 7 days. There were no secondary lesions formed and there was noevidence of spread to non-vaccinated contact chickens. The results ofthe challenge experiment are shown in Table VIII and the associatedserological findings in Table IX.

                                      TABLE VIII                                  __________________________________________________________________________    Protection of Chickens Mediated by H5 Expressed in Fowl Pox                   Challenge    Age of                                                                              Protection                                                                             Virus detected                                    Virus Vaccine                                                                              Chickens                                                                            Sick/Dead/Total                                                                        Trachea                                                                            Cloaca                                       __________________________________________________________________________    Ty/Ireland                                                                          Fowl Pox-H5                                                                          2-day  0/0/10   0/10                                                                               0/10                                        (H5N8)                                                                              (vFP-11)                                                                             5 weeks                                                                              0/0/5    0/5  0/5                                               Inactivated                                                                          2-day  0/0/9    0/9  0/9                                               H5N2   5 weeks                                                                              0/0/5    0/5  0/5                                               Fowl Pox                                                                             2-day 10/9/10   2/6  3/6                                               control                                                                              5 weeks                                                                              4/3/5    0/5  4/5                                               None   2-day 10/9/10   2/7  5/7                                                      2-day*                                                                               2/1/2    2/2  2/2                                                      5 weeks                                                                              2/2/5    0/5  1/5                                         Ck/Penn                                                                             Fowl Pox-H5                                                                          2-day  0/0/10   8/10                                                                               0/10                                        (H5N2)                                                                              (vFP-11)                                                                             5 weeks                                                                              0/0/6    5/6  2/6                                               Inactivated                                                                          2-day  0/0/8    2/8  0/8                                               H5N2   5 weeks                                                                              0/0/5    3/5  0/5                                               Fowl Pox                                                                             2-day 10/1/10  10/10                                                                              10/10                                              control                                                                              5 weeks                                                                              5/0/5    5/5  5/5                                               None   2-day  9/3/9    9/9  9/9                                                      2-day*                                                                               2/2/2    2/2  2/2                                                      5 weeks                                                                              5/2/5    5/5  5/5                                         __________________________________________________________________________     *Four nonvaccinated birds were housed and raised with the Fowl PoxH5 grou     of 10 birds to test for spread of Fowl PoxH5.                            

                                      TABLE IX                                    __________________________________________________________________________    Serological Response Induced by Inoculation with vFP-11 or an Inactivated     Influenza Virus Vaccine                                                                         HI titers to:.sup.(a)                                                                          Neutralization of Infectivity              Challenge    Age of                                                                             Ty/Ireand                                                                              Ck/Penn Ty/Ireland                                                                             Ck/Penn                           Virus Vaccine                                                                              Chickens                                                                           Post-1                                                                            Post-2                                                                             Post-1                                                                            Post-2                                                                            Post-1                                                                            Post-2                                                                             Post-1                                                                            Post-2                        __________________________________________________________________________    Ty/Ireland                                                                          Fowl Pox-H5                                                                          2-day                                                                               15.sup.(c)                                                                        156 <   65   70  2.500                                 (H5N8)       5 weeks                                                                            100  480 <   20  160  10.000                                      Inactivated                                                                          2-day                                                                               30  70   30 50   65  1.000                                       H5N2   5 weeks                                                                            350  600 180 200 240  2.500                                       Fowl Pox                                                                             2-day                                                                              <    160.sup.(1)(b)                                                                    <   20  <   300.sup.(1)                                  control                                                                              5 weeks                                                                            <   1280.sup.(2)                                                                       <   60  <    10.000.sup.(2)                              None   2-day                                                                              <    80.sup.(1)                                                                        <   20  <   300.sup.(1)                                         5 weeks                                                                            <   2000.sup.(3)                                                                       <   60  <    70.sup.(3)                            Ck/Penn/                                                                            Fowl Pox-H5                                                                          2-day                                                                               15  600 <   90           <   70                            (H5N2)       5-weeks                                                                             80 2500 <   300          <   2.500                               Inactivated                                                                          2-day                                                                               60  300  20 70            10 400                                 H5N2   5 weeks                                                                            300  500 100 200          150 1.500                               Fowl Pox                                                                             2-day                                                                              <    60.sup.(6)                                                                        <   120          <   40                                  control                                                                              5 weeks                                                                            <    90  <   140          <   40                                  None   2-day                                                                              <    60.sup.(6)                                                                        <   160          <   25                                         5 weeks                                                                            <    160.sup.(3)                                                                       <   150          <   70                            __________________________________________________________________________     .sup.(a) The 5 week old birds were bled before vaccination and tested in      HI and neutralization tests, none contained detectable antibody levels an     the results are not shown. The 2day old chickens were bled at 6 weeks         postvaccination (Post1) and the 5 week old birds were bled at 5 weeks         postvaccination (Post1): both groups were bled 2 weeks after challenge        (Post2). The FIGS. are the mean antibody titers from the same groups of       chickens described in Table 1.                                                .sup.(b) The numbers in parenthesis are those that survived challenge.        < = less than 10                                                              .sup.(c) Hemagglutamation inhibition (HI) tests were done in microtiter       plates using receptordestroying-enzyme-treated sera. 4 HA units of TY/Ire     virus, and 0.5% chicken erthrocytes as described in Palmer et al.. Immun.     Series No. 6 51-52 , U.S. Dept. Health, Education and Welfare (1975).         Neutralization of infectivity assays were done by incubating 10 EID.sub.5     of Ty/Ire virus with dilutions of sera for 30 minutes at room temperature     followed by inoculation of aliquots into embryonated eggs. Virus growth       was determined by hemagglutination assays after incubation of eggs for 2      days at 33° C..                                                   

Chickens inoculated with the fowlpox-H5 recombinant (vFP-11) or theinactivated H5N2 influenza vaccine in adjuvant were protected fromchallenge with the homologous Ty/Ire (H5N8) influenza virus and with therelated but distinguishable Ck/Penn (H5N2) influenza virus. In contrastthe majority of birds inoculated with parental FPV or that received novaccines had clinical signs of highly pathogenic influenza includingswelling and cyanosis of the face and comb, hemorrhage of the legs andparalysis. The majority of these birds died. The vaccinated birds didnot shed detectable levels of Ty/Ire but did shed Ck/Penn.

Both the inactivated and recombinant vaccines induced HI andneutralizing antibodies to Ty/Ire but the levels of antibody induced bythe fowlpox-H5 recombinant, vFP-11, prior to challenge did not inhibitHA or neutralize the heterologous Ck/Penn H5. Regardless, the chickenswere protected from challenge with both Ty/Ire and Ck/Penn influenzaviruses.

Immunity to H5 influenza induced by the vFP-11 vaccination lasted for atleast 4 to 6 weeks and was cross-reactive. To investigate further theduration and specificity of the response, a group of 4 week old chickenswas inoculated in the wing web with vFP-11 as described previously andchallenged at monthly intervals with the cross reactive Ck/Penn virus.Again, no HI antibodies were detectable prior to challenge. Nonetheless,birds were protected beyond four months.

The H5 expressed by vFP-11 also induces a protective immune response inturkeys. Outbread white turkeys were vaccinated at 2 days and 4 weeks ofage by wing-web inoculation as previously described. The results areshown in Table X.

                                      TABLE X                                     __________________________________________________________________________    Protection of Turkeys Mediated by H5-HA Expressed in vFP-11                                                       Neutralizing                                                  Virus   HI antibody                                                                           antibody log.sub.(10)                            Age  Protection                                                                            Detection                                                                             Ty/Ire  to Ty/Ire                                 Vaccine                                                                              of birds                                                                           sick/dead/total                                                                       trachea                                                                           cloaca                                                                            Post 1                                                                            Post-2                                                                            Post-1                                                                            Post-2                                __________________________________________________________________________    vFP-11 2-day                                                                              1/1/5   5/5 3/5 <10 160 <1  4.32                                  recombinant                                                                          4-week                                                                             2/1/6   2/6 0/6 <10 640 1.05                                                                              4.16                                  Contact                                                                              2-day                                                                              2/2/2   2/2 2/2 <10 dead                                                                              <1  dead                                  controls                                                                             4-week                                                                             2/2/2   2/2 2/2 <10 dead                                                                              <1  dead                                  __________________________________________________________________________

Significant survival against challenge with the homologous Ty/Ire viruswas observed with both age groups.

Non-vaccinated contact control birds were housed with the vaccinatedbirds to test for spread of the recombinant virus. These birds did notsurvive challenge.

EXAMPLE 11 CONSTRUCTION OF FOWLPOX VIRUS FP-1 RECOMBINANT vFP 12EXPRESSING CHICKEN INFLUENZA NUCLEOPROTEIN (NP) GENE

Plasmid pNP 33 contains a cDNA clone of the influenza virusChicken/Pennsylvania/1/83 nucleoprotein gene (NP). Only the 5' and 3'ends of the approximately 1.6 Kbp NP gene have been sequenced. NP wasmoved from pNP 33 into Sma I digested pUC 9 as a blunt ended 5' ClaI-Xho I 3' fragment, with the pUC 9 Eco RI site at the 3' end,generating pRW 714. The translational initiation codon (ATG) of NPcontains the following underlined Aha II site: ATGGCGTC. The vaccinia H6promoter, previously described, was joined to the NP with a doublestranded synthetic oligonucleotide. The synthetic oligonucleotidecontained the H6 sequence from the Eco RV site to its ATG and into theNP coding sequence at the Aha II site. The oligonucleotide wassynthesized with Bam HI and Eco RI compatible ends for insertion intopUC 9 generating pRW 755. Starting at the Bam HI compatible end, withthe ATG underlined, the sequence of the double stranded syntheticoligonucleotide is: ##STR1##

The Aha II linear partial digestion product of pRW 755 was isolated andrecut with Eco RI. The pRW 755 fragment containing a single Aha II cutat the ATG and recut with Eco RI was isolated, treated with phosphatase,and used as a vector for the pRW 714 digestion product below.

The isolated Aha II linear partial digestion product of pRW 714 wasrecut with Eco RI. An approximately 1.6 Kbp Aha II-Eco RI isolatedfragment, containing the NP coding sequence, was inserted into the abovepRW 755 vector generating pRW 757. The complete H6 promoter was formedby adding the sequences upstream (5') of the Eco RV site. The plasmidpRW 742B (described in Example 4) had the H6 sequence downstream (3') ofthe Eco RV site removed along with sequences through to pUC 9's Nde Isite. The pRW 742B Eco RV-Nde I fragment, treated with phosphatase, wasused as a vector for the pRW 757 fragment below. The isolated linearpartial Eco RV digestion product of pRW 757 was re-isolated after Nde Idigestion; this fragment contains the H6 promoter from the Eco RV sitethrough NP to the pUC 9 Nde I site. The pRW 757 fragment was insertedinto the pRW 742B vector to form pRW 758. The Eco RI fragment from pRW758, containing the entire H6 promoted NP, was blunt ended with theKlenow fragment of DNA polymerase I and inserted into the pRW 731.13Hinc II site generating pRW 760. The pRW 731.13 Hinc II site is the FP-1locus used in Example 6 for construction of vFP-6 and vFP-7.

Using fowlpox FP-1 as the rescuing virus, plasmid pRW 760 was used in anin vitro recombination test. Progeny plaques were assayed and plaquepurified using in situ plaque hybridization. Expression of the gene hasbeen confirmed by immune precipitation studies using a goat polyclonalanti-NP antiserum. The size of the protein specifically precipitatedfrom a lysate of vFP-12 infected CEF cells was approximately 55 KD,within the published range of influenza virus nucleoproteins.

EXAMPLE 12 PRODUCTION OF A FOWLPOX VIRUS DOUBLE RECOMBINANT vFP-15EXPRESSING THE AVIAN INFLUENZA NUCLEOPROTEIN (NP) AND HEMAGGLUTININ (HA)GENES

The hemagglutinin (HA) gene from A/Tyr/Ire/1378/83 was previouslydescribed in the construction of vFP-11 (example 9). In making a doublerecombinant the HA gene was first moved to locus f8 previously definedin the construction of vFP-8 using plasmid pRW 731.15.

The plasmid used in the construction of vFP-11 was pRW 759. Thehemagglutinin gene linked to the H6 promoter was removed from thisplasmid by a Pst I partial digest. This fragment was then blunt-endedwith the Klenow fragment of DNA polymerase I and inserted into theblunt-ended Bam HI site of pRW 731.15 to create pRW 771.

Plasmid pRW 771 was then used in an in vitro recombination test usingvFP-12 as the rescuing virus. The vFP-12 recombinant virus contains thenucleoprotein gene linked to the H6 promoter at locus f7 defined inplasmid pRW 731.13. Recombinant plaques now containing both insertionswere selected and plaque purified by in situ hybridization and surfaceexpression of the hemagglutinin confirmed by aProtein-A-Beta-galactosidase linked immunoassay. Expression of bothgenes was confirmed by immune precipitation from the double recombinantvirus, vFP-15, infected cell lysates.

EXAMPLE 13 CONSTRUCTION OF RECOMBINANT CANARYPOX VIRUSES

The following example demonstrates identification of four non-essentialinsertion loci in the canarypox genome and the construction of fourrecombinant canarypox viruses vCP-16, vCP-17, vCP-19 and vCP-20.

The recombinant canarypox vCP-16 was constructed as follows.

A 3.4 Kbp Pvu II canarypox DNA fragment was cloned into pUC 9 to producepRW 764.2. A unique Eco RI site was found asymmetrically located withinthe fragment with a short arm of 700 bp and a long arm of 2.7 Kbp. Theplasmid was digested with Eco RI and blunt-ended using the Klenowfragment of DNA polymerase I. The blunt-ended H6/rabies G gene was thenligated into this site and used to transform E. coli. The resultingplasmid pRW 775 was used in an in vitro recombination test. Progenyplaques positive on an immunoscreen were selected and plaque purified.The resulting recombinant was designated vCP-16 and the insertion locusas C3.

The plasmid pRW 764.2 used in the construction above also contained aunique Bgl II site approximately 2.4 Kbp from the Eco RI site. Using thesame cloning strategy the H6/rabies G gene was ligated into plasmid pRW764.2 at this site to produce pRW 774. This plasmid was used in theconstruction of recombinant vCP-17 with the insertion locus designatedas C4.

Plasmid pRW 764.5 contains an 850 bp Pvu II fragment of canarypox DNAwith a unique Bgl II site assymmetric within the fragment 400 bp fromone terminus. Using the same cloning strategy previously described therabies G gene linked to the H6 promoter was inserted at this site toproduce pRW 777. The stable recombinant virus produced was designatedvCP-19 and the insertion locus C5.

Plasmid pRW 764.7 contains a 1.2 Kbp Pvu II fragment with a unique BglII site 300 bases from one terminus. The plasmid was digested with BglII and blunt-ended with the Klenow fragment of DNA polymerase I. Theblunt-ended 11K promoted Lac Z gene was inserted to produce plasmid pRW778. The stable recombinant virus produced using this plasmid wasdesignated vCP-20 and the insertion locus designated C6.

EXAMPLE 14 CONSTRUCTION OF FOWLPOX VIRUS RECOMBINANT vFP-29 EXPRESSINGTHE FUSION PROTEIN OF NEWCASTLE DISEASE VIRUS

Plasmid pNDV 108, the cDNA clone of the fusion gene of NDV Texas Strain,consisted of an Hpa I cDNA fragment of approximately 3.3 Kbp containingthe fusion protein coding sequence as well as additional NDV codingsequences cloned into the Sca I site of pBR 322. Steps in the productionof the insertion plasmid are described below.

(1) Creation of plasmid pCE 11

An FPV insertion vector, pCE 11, was constructed by insertingpolylinkers at the Hinc II site of pRW 731.13 (designated as locus f7).pRW 731.13 contains a 5.5 Kbp Pvu II fragment of FP-1 DNA. Anonessential locus was previously defined at the Hinc II site by theconstruction of the stable recombinant vFP-6 previously described inExample 6. The polylinkers inserted at the Hinc II site contain thefollowing restriction enzyme sites: Nru I, Eco RI, Sac I, Kpn I, Sma I,Bam HI, Xba I, Hinc II, Sal I, Acc I, Pst I, Sph I, Hind III and Hpa I.

(2) Creation of plasmid pCE 19

This plasmid is a further modification of pCE 11, in which the vacciniavirus transcriptional stop signal ATTTTTNT (L. Yuen and B. Moss, J.Virology 60, 320-323 [1986]) (where N in this case is an A) has beeninserted between the Sac I and Eco RI sites of pCE 11 with theconsequent loss of the Eco RI site.

(3) Insertion of NDV coding sequences

A 1.8 Kbp gel-purified Bam HI fragment containing all but 22 nucleotidesfrom the 5' end of the fusion protein gene was inserted into the Bam HIsite of pUC 18 to form pCE 13. This plasmid was digested with Sal Iwhich cuts in the vector 12 bases upstream of the 5' end of the codingsequence. The ends were filled in with the Klenow fragment of DNAPolymerase I and the plasmid further digested with Hind III which cuts18 bases upstream of the Sal I site. A gel purified 146 bp Sma I-HindIII fragment containing the vaccinia virus H6 promoter previouslydescribed in preferred embodiments as well as polylinker sequences ateach termini was ligated to the vector and transformed into E. colicells. The resulting plasmid was designated pCE 16.

In order to align the initiating ATG codon of the NDV fusion proteingene with the 3' end of the H6 promoter and to replace the 22nucleotides missing from the NDV 5' end in pCE 16, complementarysynthetic oligonucleotides were designed ending in Eco RV and Kpn Isites. The oligonucleotide sequence was 5'ATC-CGT-TAA-GTT-TGT-ATC-GTA-ATG-GGC-TCC-AGA-TCT-TCT-ACC-AGG-ATC-CCG-GTA-C3'.

The construct pCE 16 was then digested with Eco RV and Kpn I. The Eco RVsite occurs in the H6 promoter 24 bases upstream of the initiating ATG.The Kpn I site occurs in the NDV coding sequence 29 bases downstream ofthe ATG.

Oligonucleotides were annealed, phosphorylated and ligated to thelinearized plasmid and the resulting DNA used to transform E. colicells. This plasmid was designated pCE 18.

In order to insert the NDV coding sequence into an FPV insertion vector,a gel purified 1.9 Kbp Sma I-Hind III fragment of pCE 18 (cutting in thepolylinker region) was ligated to a 7.8 Kbp Sma I-Hind III fragment ofpCE 19 described above. The transcriptional stop signal occurs 16 basesdownstream of the Sma I site. The resulting plasmid was designated pCE20.

The plasmid pCE 20 was used in an in vitro recombination test usingfowlpox virus FP-1 as the rescuing virus. The resulting progeny wereplated on CEF monolayers and the plaques subjected to aBeta-galactosidase linked Protein-A immunoscreen using a polyclonalanti-NDV chicken serum. Positively staining plaques were selected andsubjected to four rounds of plaque purification to achieve a homogeneouspopulation. The recombinant was designated vFP-29.

EXAMPLE 15 CONSTRUCTION OF AVIPOX VIRUS RECOMBINANTS EXPRESSING THEFELINE LEUKEMIA VIRUS (FeLV) ENVELOPE (ENV) GLYCOPROTEIN

The FeLV env gene contains the sequences which encode the p70+p15Epolyprotein. This gene was initially inserted into the plasmid pSD467vCwith the vaccinia H6 promoter juxtaposed 5' to the FeLV env gene. Theplasmid pSD467vC was derived by first inserting an 1802 bp Sal I/HindIII fragment containing the vaccinia hemagglutinin (HA) gene into apUC18 vector. The location of the HA gene was defined previously (Shida,Virology 150, 451-462, [1988]). The majority of the open reading frameencoding the HA gene product was deleted (nucleotide 443 throughnucleotide 1311) and a multiple cloning site was inserted containing theBgl II, Sma I, Pst I, and Eag I restriction endonuclease sites. Theresultant pSD467vC plasmid contains vaccinia flanking arms of 442 bpupstream of the multiple cloning site and 491 bp downstream from theserestriction sites. These flanking arms enable genetic material insertedinto the multiple cloning region to be recombined into the HA region ofthe Copenhagen strain of vaccinia virus. The resultant recombinantprogeny are HA negative.

The H6 promoter was synthesized by annealing four overlappingoligonucleotides which together comprised the complete sequencedescribed above in preferred embodiments. The resultant 132 bp fragmentcontained a Bgl II restriction site at the 5' end and a Sma I site atthe 3' end. This was inserted into pSD467vC via the Bgl II and Sma Irestriction site. The resultant plasmid was designated pPT15. The FeLVenv gene was inserted into the unique Pst I site of pPT15 which is justdownstream of the H6 promoter. The resultant plasmid was designatedpFeLV1A.

For construction of the FP-1 recombinant, the 2.4 Kbp H6/FeLV envsequences were excised from pFeLV1A by digestion with Bgl II and partialdigestion with Pst I. The Bgl II site is at the 5' border of the H6promoter sequence. The Pst I site is located 420 bp downstream from thetranslation termination signal for the envelope glycoprotein openreading frame.

The 2.4 Kbp H6/FeLV env sequence was inserted into pCE 11 digested withBam HI and Pst I. The FP-1 insertion vector pCE 11, was derived from pRW731.13 by insertion of a multiple cloning site into the nonessentialHinc II site. This insertion vector allows for the generation of FP-1recombinants harboring foreign genes in locus f7 of the FP-1 genome. Therecombinant FP-1/FeLV insertion plasmid was then designated pFeLVF1.This construction does not provide a perfect ATG for ATG substitution.

To achieve the perfect ATG:ATG construction, a Nru I/Sst II fragment ofapproximately 1.4 Kbp was derived from the vaccinia virus insertionvector, pFeLV1C. The Nru I site occurs within the H6 promoter at aposition 24 bp upstream from the ATG. The Sst II site is located 1.4 Kbpdownstream from the ATG and 1 Kbp upstream from the translationtermination signal. This Nru I/Sst II fragment was ligated to a 9.9 Kbpfragment which was generated by digestion with Sst II and by partialdigestion with Nru I. This 9.9 Kbp fragment contains the 5.5 Kbp of FP-1flanking arms, the pUC vector sequences, 1.4 Kbp of FeLV sequencecorresponding to the downstream portions of the env gene, and the5'-most sequence (approx. 100 bp) of the H6 promoter. The resultantplasmid was designated pFeLVF2. The ATG for ATG construction wasconfirmed by nucleotide sequence analysis.

A further FP-1 insertion vector, pFeLVF3, was derived from pFeLVF2 byremoving the FeLV env sequences corresponding to the putativeimmunosuppressive region (Cianciolo et al., Science 230, 453-455 [1985])(nucleotide 1548 to 1628 of coding sequence). This was accomplished byisolating a Sst II/Pst I fragment (sites described above) ofapproximately 1 Kbp from the vaccinia virus insertion vector pFeLV1D.The plasmid pFeLV1D is similar to pFeLV1C except that the env sequencescorresponding to the immunosuppressive region (nucleotide 1548 to 1628)were deleted by oligonucleotide-directed mutagenesis (Mandecki, Proc.Natl. Acad. Sci. USA 83, 7177-7181 [1987]). The 1 Kpb Sst II/Pst Ifragment lacking nucleotides 1548 to 1628 was inserted into a 10.4 KbpSst II/Pst I fragment containing the remaining H6:FeLV env gene derivedfrom pFeLVF2.

The insertion plasmids, pFeLVF2 and pFeLVF3, were used in in vitrorecombination tests with FP-1 as the rescuing virus. Progeny of therecombination were plated on CEF monolayers and recombinant virusselected by plaque hybridization on CEF monolayers. Recombinant progenyidentified by hybridization analyses were selected and subjected to 4rounds of plaque purification to achieve a homogeneous population. AnFP-1 recombinant harboring the entire FeLV env gene has been designatedvFP-25 and an FP-1 recombinant containing the entire gene lacking theimmunosuppressive region was designated vFP-32. Both recombinants havebeen shown to express the appropriate gene product byimmunoprecipitation using a bovine anti-FeLV polyclonal serum(Antibodies, Inc., Davis, Calif.). Significantly, these FP-1recombinants express the foreign FeLV env gene in the CRFK cell line(ATCC #CCL94), which is of feline origin.

For construction of the canarypox (CP) recombinants, a 2.2 Kbp fragmentcontaining the H6:FeLV env sequences was excised from pFeLVF2 bydigestion with Sma I and Hpa I. The Sma I site is at the 5' border ofthe H6 promoter sequence. The Hpa I site is located 180 bp downstreamfrom the translation termination signal for the envelope glycoproteinopen reading frame.

The 2.2 Kbp H6/FeLV env sequence was inserted in the non-essential EcoRI site of the insertion plasmid pRW764.2 following blunt-ending of theEco RI site. This insertion vector allows for the generation of CPrecombinants harboring foreign genes in locus C4 of the CP genome. Therecombinant CP insertion plasmid was then designated pFeLVCP2. Thisconstruction provides a perfect ATG for ATG substitution.

The insertion plasmid, pFeLVCP2, was used in an in vitro recombinationtest with CP as the rescuing virus. Progeny of the recombinant wereplated on CEF monolayers and recombinant virus selected by means of aBetagalactosidase linked Protein-A immunoscreen using a bovine anti-FeLVcommercial polyclonal serum (Antibodies, Inc., Davis, Calif.). Positivestaining plaques were selected and subjected to four rounds of plaquepurification to achieve a homogeneous population. A recombinantexpressing the entire FeLV env gene has been designated vCP-36.

EXAMPLE 16 CONSTRUCTION OF FOWLPOX VIRUS RECOMBINANT vFP-22 EXPRESSINGTHE ROUS ASSOCIATED VIRUS TYPE 1 (RAV-1) ENVELOPE (ENV) GENE

The clone penvRV1PT of the RAV-1 envelope gene contains 1.1 Kbp of RAV-1env DNA coding sequence cloned as a Kpn I-Sac I fragment into M13mp18.This fragment is intact at the 5' end but lacks part of the 3' sequenceand was used in the following manipulations. A gel purified 1.1 Kbp EcoRI-Pst I fragment from penvRVIPT was inserted into the Eco RI and Pst Isites of pUC 9 to form pRW 756. This plasmid was then digested with KpnI and Hind III cutting in the vector 59 bases upstream of the ATG. A 146base pair Kpn I-Hind III fragment containing the previously describedvaccinia H6 promoter was inserted to construct plasmid pCE 6.

In order to ensure that the initiating ATG of the RAV env gene wasadjacent to the 3' end of the H6 promoter with extraneous sequencesdeleted, two complementary synthetic oligonucleotides were constructedwith Eco RV and Ban II sites at the termini. The oligonucleotidesequence was 5' ATC-CGT-TAA-GTT-TGT-ATC-GTA-ATG-AGG-CGA-GCC-3'

The plasmid pCE 6 was digested with Eco RV which cuts in the H6 promoter24 bases upstream of the ATG and Ban II which cuts in the RAV env codingsequence 7 bases downstream of the ATG. The DNA segments were ligatedand used to transform E. coli cells. The resulting plasmid, pCE 7,supplied the H6 promoter and correct 5' sequence for the finalconstruction.

Clone mp19env (190), was found by restriction mapping to contain theentire RAV-1 env gene. A 1.9 Kbp Kpn I-Sac I fragment of the mp19env(190) containing the entire gene was inserted at the Kpn I and Sac Isites of pUC 18 to form pCE 3. This plasmid was digested with Hpa Iwhich cuts 132 bases downstream of the initiating ATG in the RAV-1coding sequence and Sac I which cuts at the 3' terminus of the gene. TheFPV insertion vector pCE 11 previously described was digested with Sma Iand Sac I cutting the plasmid in the polylinker region. The Hpa I-Sac Ifragment of pCE 3 was ligated with pCE 11 to form pCE 14.

The plasmid pCE 7 was then digested with Xho I and Hind III to provide a332 base pair fragment containing the H6 promoter and correct 5'sequence. Plasmid pCE 14 was digested with Hind III cutting in thepolylinker region of the vector and Xho I cutting in the codingsequence. This DNA was ligated with the Hind III-Xho I fragment obtainedfrom pCE 7 to form pCE 15, the final RAV-1 envelope gene construct.

This plasmid was used in an in vitro recombination test with fowlpoxFP-1 as the rescuing virus. Progeny of the recombination was plated onCEF monolayers and plaques screened by a Beta-galactosidase linkedProtein A immunoassay using an anti-RAV-1 polyclonal serum. Positivelystaining plaques were selected and subjected to four rounds of plaquepurification to produce a homogeneous population. The recombinantproduced was designated vFP-22. Immunoprecipitation experiments usingvFP-22 infected CEF lysates have demonstrated the specific precipitationof two proteins with apparent molecular weights of 76.5 Kd and 30 Kdcorresponding to the two gene products of the envelope gene. Noprecursor gene product was apparent.

In preliminary tests an immune response has been induced to the RAV-Ienvelope gene product in chickens inoculated with vFP-22.

EXAMPLE 17 CONSTRUCTION OF AVIPOX VIRUS RECOMBINANTS EXPRESSING THEGP51,30 ENVELOPE (ENV) GENE OF BOVINE LEUKEMIA VIRUS (BLV)

(1) Construction of pBLVF 1 and pBLVF 2

The plasmids, pBLVF 1 and pBLVF 2, contain the gp51,30 env gene of BLV.In both plasmids, the BLV env gene is under the transcriptional controlof the vaccinia virus H6 promoter and is cloned between fowlpox flankingarms (locus f7). The nucleotide sequence of the two plasmids isidentical, except at codon positions 268 and 269. (pBLVF 1 encodes aprotein containing the amino acids Arg-Ser at these two positions,whereas pBLVF 2 encodes a protein containing the amino acids Gln-Thr).

pBLVF 1 and pBLVF 2 were constructed by the following procedure. PlasmidpNS97-1, a plasmid containing the entire BLV env gene, was cut with BamH1 and partially cut with Mst II. The 2.3 Kbp fragment containing theentire gp51,30 gene was isolated on an agarose gel and the sticky endsfilled in with E. coli DNA polymerase I (Klenow fragment). Pst I linkerswere then ligated onto the ends of the fragment, which after Pst Idigestion, was ligated into the Pst I site of pTP 15 (Example 15). Thisplaces the BLV gene next to the vaccinia H6 promoter. (pTP15 containsthe vaccinia H6 promoter cloned at a nonessential locus in the vacciniagenome.

This plasmid was then cut with Eco RV and partially cut with Ava II. The5.2 Kbp fragment was isolated and the oligonucleotides5'-ATCCGTTAAGTTTGTATCGTAATGCCCAAAGAACGACG-3 and5'GACCGTCGTTCTTTGGGCATTACGATACAAACTTAACGGAT-3' used to recircularize theplasmid. This removes unnecessary bases between the BLV gene and the H6promoter.

The resulting plasmid was cut with Pst I and partially cut with Bgl IIand the 1.7 Kbp fragment containing the H6 promoted-BLV gene cloned intothe Bam HI-Pst I site of pCE 11, the fowlpox virus insertion vectorpreviously described using locus f7. This places the H6 promoted-BLVgene between fowlpox flanking arms. This plasmid was designated pBLVF 1.

An identical procedure was used to construct pBLVF 2, with the exceptionthat an additional in vitro mutagenesis step was performed beforecloning the H6 promoted-BLV gene into pCE 11. This mutagenesis wasperformed by the following procedure. Plasmid pNS97-1 was cut with Xma Iand partially cut with Stu I. The 5.2 Kbp fragment was isolated and theoligonucleotides 5'-CCGGGTCAGACAAACTCCCGTCGCAGCCCTGACCTTAGG-3' and5'-CCTAAGGTCAGGGCTGCGACGGGAGTTTFTCTGAC-3' used to recircularize theplasmid. This changes the nucleotide sequence of codons 268 and 269 fromCGC-AGT to CAA-ACT.

(2) Construction of Recombinant Viruses

The plasmids pBLVF 1 and pBLVF 2 were used in an in vitro recombinationtest using FP-1 as the rescuing virus. Recombinant progeny was selectedby in situ plaque hybridization and when the population was judged aspure by this criteria plaques were screened in anBeta-galactosidase-Protein A immunoassay using a BLV gp specificmonoclonal antibody preparation. Both recombinants vFP 23 and vFP 24produced from plasmid pBLVF 1 and pBLVF 2 respectively showed positivestaining in the immunoscreen indicating that an immunologicallyrecognizable glycoprotein was expressed on the infected cell surface.

The plasmids, pBLVK 4 and pBLVK 6 contain the BLV env gp51,30 gene andthe BLV gp51,30 cleavage minus gene, respectively. Both genes are clonedinto the unique Eco RI site of pRW 764.2 (locus C3) (pRW 764.2 isdescribed in Example 13) and are under the transcriptional control ofvaccinia H6 promoter.

The plasmids were derived by the following procedure: pBLVF 1 and pBLVF2 were cut with the restriction enzyme Hind III the oligonucleotide BKL1 (AGCTTGAATTCA) was cloned into this site, thereby generating an Eco RIsite 3' to the BLV gene. Since there is also an Eco RI site 5' to theBLV gene, these plasmids (pBLVK 1 and pBLVK 2) were cut with Eco RI andthe fragment containing the H6 promoted-BLV gene was cloned into the EcoRI site of pRW 764.2. The resulting plasmids were designated pBLVK 4 andpBLVK 6, respectively. These plasmids were used in an in vitrorecombination test with canarypox as the rescuing virus. Recombinantswere selected and purified on the basis of surface expression of theglycoprotein as detected in an immunoassay. The recombinants weredesignated vCP 27 and vCP 28 from plasmids pBLVK 4 and pBLVK 6,respectively.

Fowlpox recombinants vFP23 and vFP24 have been inoculated into sheep andbovines by a variety of routes. Animals were given two inoculations, thesecond at 45 days after the first. Serum samples were taken 5 weeksafter the first inoculation and two weeks after the second inoculation.Antibody to gp51 was measured in a competitive ELISA test and the titerexpressed as the reciprocal of the serum dilution giving a 50% reductionof competition. The results are shown in Table XI.

None of the species tested showed a detectable immune response after theprimary inoculation. Both sheep and bovines showed a significantantibody rise after the secondary inoculation.

                  TABLE XI                                                        ______________________________________                                        Inoculation of Sheep and Bovines with vFP23 and vFP24                                    Dose and Route                                                                              ELISA   Titer                                        Animal   Virus   1° 2°                                                                             1°                                                                           2°                            ______________________________________                                        Bovine                                                                              B56    FP-1    10.sup.8 + 10.sup.8a                                                                  10.sup.8 + 10.sup.8                                                                   0      0                                       B59    FP-1    ID      subcut. 0      0                                 Sheep M89    FP-1                    0      0                                       M91    FP-1                    0      0                                 Bovine                                                                              B62    vFP-23  10.sup.8 + 10.sup.8                                                                   10.sup.8 + 10.sup.8                                                                   0     .sup. 200.sup.b                          B63    vFP-23  ID      subcut. 0      80                                Sheep M83    vFP-23                  0      80                                      M84    vFP-23                  0     500                                      M85    vFP-23                  0     100                                Bovine                                                                              B52    vFP-24  10.sup.8 + 10.sup.8                                                                   10.sup.8 + 10.sup.8                                                                   0     200                                      B53    vFP-24  ID      subcut. 0      60                                Sheep M87    vFP-24                  0     200                                      M92    vFP-24                  0      20                                      M93    vFP-24                  0      20                                ______________________________________                                         .sup.a Intradermal injections were at two points                              .sup.b Titer expressed as the reciprocal of the dilution giving 50%           competition                                                              

EXAMPLE 18 CONSTRUCTION OF FOWLPOX VIRUS FP-1 RECOMBINANT VFP-26EXPRESSING THE INFECTIOUS BRONCHITIS VIRUS MASS 41 MATRIX GENE.

Plasmid pIBVM63 contains an infectious bronchitis virus (IBV) cDNA cloneof the Mass 41 strain matrix gene. An 8 Kbp Eco RI fragment of pIBVM63contains the matrix gene with the peplomer gene upstream (5') andfurther upstream there is an Eco RV site. Plasmid pRW 715 has an Eco RIlinker joining the two Pvu II sites of pUC 9. The 8 Kbp Eco RI fragmentfrom pIBVM63 was inserted into the pRW 715 Eco RI site generatingpRW763. Plasmid pRW 776 was created to delete the 5' Eco RI site in pRW763, leaving a unique Eco RI site downstream (3') of the matrix gene.The isolated linear Eco RI partial digestion product of pRW 763 wasrecut with Eco RV. The largest fragmemt was isolated, blunt ended withthe Klenow fragment of DNA polymerase I and self ligated generating pRW776. The construct pRW 776 has the complete IBV peplomer and matrixgenes followed by a single Eco RI site.

Only the 5' and 3' ends of the approximately 0.9 Kbp matrix gene havebeen sequenced. The 5' sequence of the matrix gene, starting at thetranslational initiation condon (ATG), contains the following underlinedRsa I site: ATGTCCAACGAGACAAATTGTAC. The previously describe H6 promoterwas joined to the matrix gene with a synthetic oligonucleotide. Thesynthetic oligonucleotide contained the H6 sequence from its Eco RV siteto the ATG and into the matrix coding sequence through the first Rsa Isite. The oligonucleotide was synthesized with Bam HI and Eco RIcompatible ends for insertion into pUC 9 generating pRW 772. The Eco RIend is 3' to the Rsa I site. Starting at the Bam HI compatible end, withthe ATG underlined, the sequence of the double stranded syntheticoligonucleotide is: ##STR2##

The Rsa I linear partial digestion product of pRW 772 was isolated andrecut with Eco RI. The pRw 772 fragment containing a single cut at theabove Rsa I site and recut with Eco RI was isolated, treated withphosphatase, and used as a vector for the pRW 776 digestion productbelow.

The isolated Rsa I linear partial digestion product of pRW 776 was recutwith Eco RI. Eco RI is just beyond the 3' end of the matrix gene. Anapproximately 0.8 Kbp Rsa I-Eco RI isolated fragment, containing thematrix coding sequence from the above Rsa I site, was inserted into theabove pRW 772 vector generating pRW 783. The complete H6 promoter wasformed by adding sequences 5' of the Eco RV site. The H6 promoter 5' endwas a Hinf I site blunt ended into the pUC 9 Sal I site creating an EcoRI site; 5' of the H6 promoter is the pUC 9 Hind III site. The HindIII-Eco RV fragment containing the 5' H6 promoter was inserted betweenthe pRW 783 Hind III and Eco RV sites generating pRW 786. The pRW 786Eco RI fragment, containing the complete H6 promoted matrix gene, wasblunt ended with Klenow fragment of DNA polymerase I and inserted intothe blunt ended Bam H1 site of pRW 731.15(locus f8) generating pRW 789.The pRW 731.15 Bam HI site is the FP-1 locus used in Example 6 forconstruction of vFP-8.

Plasmid pRW 789 was used in the construction of vFP-26. Recombinantplaques were selected and processed by in situ plaque hybridization.

In preliminary tests an immune response has been induced to the IBVmatrix protein in chickens inoculated with vFP-26.

EXAMPLE 19 CONSTRUCTION OF FOWLPOX VIRUS FP-1 RECOMBINANT vFP-31EXPRESSING INFECTIOUS BRONCHITIS VIRUS (IBV) PEPLOMER

The infectious bronchitis virus (IBV) Mass 41 cDNA clone pIBVM 63 andits subclone, pRW 776, have been described for the vFP-26 constructionin Example 18. Subclone pRW 776 contains the 4 Kbp IBV peplomer genefollowed by the matrix gene with a unique Eco RI site at the 3' end.Only the 5' and 3' ends of the approximately 4 Kbp IBV peplomer genehave been sequenced. A unique Xba I site separates the two genes. The 5'end of the peplomer gene, starting at the translational initiation codon(ATG), contains the following underlined Rsa I site:ATGTTGGTAACACCTCTTTTACTAGTGACTCTTTTGTGTGTAC. The previously described H6promoter was joined to the peplomer gene with a syntheticoligonucleotide. The synthetic oligonucleotide contains the H6 promotersequence from its Nru I site to ATG and into the peplomer codingsequence through its first Rsa I site. The oligonucleotide wassynthesized with Bam HI nd Eco RI compatible ends for insertion into pUC9 generating pRW 768. The Eco RI end is 3' of the Rsa I site. Startingat the Bam HI compatible end, with the ATG underlined, the sequence ofthe double stranded synthetic oligonucleotide is: ##STR3##

The pRW 768 isolated linear partial Rsa I digestion product was recutwith Eco RI. The pRW 768 fragment containing a single cut at the aboveRsa I site and recut with Eco RI was isolated, treated with phosphatase,and used as a vector for the pRW 776 digestion product below.

The pRW 776 isolated linear partial Rsa I digestion product was recutwith Eco RI. The 5 Kbp pRW 776 fragment containing a single cut at theabove Rsa I site to the Eco RI site was isolated; the fragment containsIBV sequences from the above peplomer Rsa I site to the Eco RI site atthe 3' end of the matrix gene. Insertion of the pRW 776 fragment intothe above pRW 768 vector generated pRW 788 . The matrix gene was removedat the Xba I site noted above. The 5' H6 promoter was added at the Nru Isite by insertion of the 4 Kbp pRW 788 Nru I-Xba I blunt ended fragmentinto the pRW 760 Nru I-Bam HI blunt ended vector generating pRW 790. Thevector pRW 760 is described in Example 11; briefly, it is vaccinia H6promoted influenza nucleoprotein flanked by the nonessential FP-1 locusf7. The pRW 760 vector was made by removing the 3' H6 sequences from theNru I site through the end of the nucleoprotein at Bam HI. pRW 790 is H6promoted IBV peplomer in the pRW 731.13 Hinc II site. Recombination ofthe donor plasmid pRW 790 with FP-1 resulted in vFP-31.Immunoprecipitation experiments using CEF lysates prepared from vFP-31infected cells have demonstrated specific precipitation of a smallamount of precursor protein with a molecular weight or approximately 180Kd and of the clevage product of 90 Kd.

EXAMPLE 20 CONSTRUCTION OF FOWLPOX VIRUS FP-1 RECOMBINANT vFP-30EXPRESSING HERPES SIMPLEX VIRUS gD

The herpes simplex virus (HSV) type 1 strain KOS glycoprotein D gene(gD) was cloned into the pUC 9 Bam HI site as a 5' Bam HI linked Hpa IIto 3' Bam HI linked Nru I fragment; the 5' end is next to the pUC 9 PstI site. The 5' sequence of HSV gD, starting at the translationalinitiation condon (ATG), contains the following underlined Nco I site:ATGGGGGGGGCTGCCGCCAGGTTGGGGGCCGTGATTTTGTTTGTCGTCATAGTGGGCCT-CCATGG. Thepreviously described vaccinia H6 promoter was joined to the HSV gD genewith a synthetic oligonucleotide. The synthetic oligonucleotide containsthe 3' portion of the H6 promoter from Nru I to ATG into the gD codingsequence through the Nco I site. The oligonucleotide was synthesizedwith a 5' Pst I compatible end. The gD clone in pUC9 was cut with Pst Iand Nco I, and the 5' HSV sequence removed, for replacement with thesynthetic oligonucleotide resulting in pRW 787. The sequence of thedouble stranded synthetic oligonucleotide is: ##STR4##

Digestion of pRW 787 with Nru I and Bam H1 generates an approximately1.3 Kbp fragment containing the 3' H6 promoter, from the Nru I site,through the HSV gD coding sequence to the Bam HI site. The pRW 760vector, cut with Nru I and Bam H1, has been described in Example 11.Insertion of the 1.3 Kbp fragment into the pRW 760 vector generated pRW791. The pRW 791 vector contains the complete vaccinia H6 promoted HSVgD gene in the nonessential FP-1 Hinc II site in pRW 731.13. (locus f7).

Recombination of the donor plasmid pRW 791 with FP-1 resulted in vFP-30.Surface expression of the glycoprotein was detected in recombinantplaques using a Protein-A-Beta-galactosidase linked immunoassay andHSV-1 specific sera.

EXAMPLE 21 USE OF ENTOMOPOX PROMOTERS FOR REGULATION OF EXPRESSION OFFOREIGN GENES IN POXVIRUS VECTORS

(a) Background. Poxviruses of insects (entomopox) are currentlyclassified in the subfamily Entomopoxvirinae which is further subdividedinto three genera (A, B, and C) corresponding to entomopoxvirusesisolated from the insect orders Coleoptera, Lepidoptera, and Orthopterarespectively. Entomopox viruses have a narrow host range in nature, andare not known to replicate in any vertebrate species.

The entomopox virus used in these studies was originally isolated frominfected Amsacta moorei (Lepidoptera: arctildae) larvae from India.(Roberts and Granados, J. Invertebr. Pathol. 12, 141-143 [1968]). Thevirus, designated AmEPV, is the type species for genus B.

Wild-type AmEPV was obtained from Dr. R. Granados (Boyce ThompsonInstitute, Cornell University) as infectious hemolymph from infectedEstigmene acrea larvae. The virus was found to replicate in aninvertebrate cell line, IPLB-LD652Y, derived from ovarial tissues ofLymantria dispar (gypsy moth). (described by Goodwin et. al., In Vitro14, 485-494 [1978]). The cells were grown in IPL-528 media supplementedwith 4% fetal calf and 4% chicken sera at 28° C.

The wild-type virus was plaque assayed on LD652Y cells and one plaque,designated V1, was selected for subsequent experiments. This isolateproduces numerous occlusion bodies (OBs) in the cytoplasm of theinfected cells late in the infectious cycle.

(b) Promoter Identification. The identification and mapping of an AmEPVpromoter was accomplished as follows. Total RNA from late infectedLD652Y cells (48 hr. post infection) was isolated and used to make32P-labelled, first strand cDNA. The cDNA was then used to probe blotscontaining restriction digests of the AmEPV genome. This Southern blotdetected a strong signal on a 2.6 kb Cla I fragment, indicating that thefragment encoded a strongly expressed gene. The fragment was cloned intoa plasmid vector and its DNA sequence determined.

Analysis of the sequence data revealed an open reading frame capable ofencoding a 42 Kd polypeptide. In vitro translation of the total RNA at48 hr. post infection and separation of the products by SDS-PAGErevealed a polypeptide of approximately 42 Kd.

(c) Construction of a recombinant vaccinia virus with expression of aforeign gene under the control of the entomopox promoter. In order todetermine if an entomopox promoter would function in a vertebratepoxvirus system, the following plasmid was constructed. Anoligonucleotide was chemically synthesized which contained the 107 bases5' of the 42K gene translational start signal (hereafter referred to asthe AmEPV 42K promoter) flanked by a Bgl II site at the 5' end and thefirst 14 bases of the hepatitis B virus pre-S2 coding region, whichterminates in an Eco RI site, at the 3' end. The AmEPV 42K promotersequence is described below. ##STR5##

The AmEPV 42K promoter was ligated to the hepatitis B virus surfaceantigen (HBVsAg) as follows. A pUC plasmid was constructed containingthe hepatitis B virus surface antigen and pre-S2 coding region (type aywdescribed by Galibert et. al., Nature 281, 646-650 [1979]) flanked byvaccinia virus arms in the non-essential region of the vaccinia virusgenome which encodes the hemagglutinin (HA) molecule (HA arms describedin Example 15; HA region described by Shida, Virology 150, 451-462[1986]). The oligonucleotide described above was inserted into thisplasmid using the unique EcoR I site in the HBVsAg coding region and aunique Bgl II site in the HA vaccinia arm. The resulting recombinantvaccinia virus was designated vP 547.

Expression of the inserted HBVsAg coding sequence under the control ofthe entomopox 42K promoter was confirmed using an immunoassay.Equivalent cultures of the mammalian cell line BSC-40 were infected withparental vaccinia virus or recombinant vP 547. At 24 hourspost-infection cells were lysed and the lysate applied in serialdilutions to a nitrocellulose membrane. The membrane was first incubatedwith a goat anti-HBV serum and then with ¹²⁵ I-Protein A. After washing,the membrane was exposed to X-ray film. Positive signals were detectedin vP 547 infected cultures but not in parental virus infected cultures,indicating recognition of the AmEPV 42K promoter by vaccinia virus inmammalian cells.

The above results were verified using an Ausria assay (see Example 1 fordetails) to detect HBVsAg in infected mammalian cells. Vaccinia virusrecombinants containing the HBsAg gene coupled to the AmEPV42K orvaccinia virus H6 promoter were used to infect BSC-40 cells and thelevel of expression of sAg assayed by the Ausria test. As presented inTable XII, the data shows that the level of expression of HBsAg usingthe 42K promoter was significant.

                  TABLE XII                                                       ______________________________________                                        Expression of HBVsAg in Recombinant Vaccinia Virus                                                      Ausria                                              Recombinant Virus                                                                              Promoter P/N Ratio                                           ______________________________________                                        vP410            Control   1.0                                                vP481            H6       24.3                                                vP547            42K      44.9                                                ______________________________________                                    

Further experiments were conducted to ascertain the temporal nature ofthe regulation of the AmEPV 42K promoter in a vertebrate poxvirusbackground. Equivalent cultures of BSC-40 cells were infected with vP547 in the presence or absence of 40 ug/ml of cytosine arabinoside, aninhibitor of DNA replication which therefore blocks late viraltranscription. Levels of expression at 24 hours post-infection wereassayed in an Ausria test. The results indicated that the 42K promoterwas recognized as an early promoter in a vaccinia virus replicationsystem.

Note that the use of the AmEPV 42K promoter for the expression offoreign genes in a mammalian system is clearly distinct from the use ofthe Autographa californica NPV polyhedrin promoter for gene expressionin invertebrate systems (Luckow and Summers, Biotechnology 6, 47-55[1988]). The polyhedrin promoter is not recognized by thetranscriptional apparatus in mammalian cells (Tjla et. al., Virology125, 107-117 [1983]). The use of the AmEPV 42K promoter in mammaliancells represents the first time an insect virus promoter has beenutilized for the expression of foreign genes in a non-insect viralvector in non-invertebrate cells.

In order to determine whether avipox viruses would also recognize the42K entomopox promoter, the following experiment was performed.Identical cultures of CEF cells were inoculated at 10 pfu per cell witheither fowlpox virus, canarypox virus or vaccinia virus, andsimultaneously transfected with 25 ug of one of the followingplasmids 1) plasmid 42K.17 containing the HBV pre-S₂ +sAg codingsequence linked to the 42K promoter or 2) plasmid pMP15.spsP containingthe identical HBVsAg coding sequence linked to the vaccinia virus H6promoter previously described. After 24 hours the cultures were frozen,the cells lysed and the lysate analyzed for the presence of HBVsAg usingan Ausria test (see Example 1).

The results shown in Table XIII should be viewed in a qualitative sense.They indicate that the transcriptional apparatus of both fowlpox andcanarypox is able to recognize the 42K promoter and allow transcriptionof the linked HBVsAg coding sequence. Although levels of expression arelower than those obtained with the vaccinia virus H6 promoter, levelsare well above background levels obtained with the negative controls.

                  TABLE XIII                                                      ______________________________________                                        Recognition of 42K Entomopox                                                  Promoter by Avipox Viruses                                                    Virus          Promoter P/N Ratio                                             ______________________________________                                        Fowlpox        42K      39.1                                                                 H6       356.8                                                 Canarypox      42K      90.2                                                                 H6       222.2                                                 Vaccinia       42K      369.4                                                                H6       366.9                                                 None           42K      7.8                                                   None           H6       7.2                                                   Vaccinia       --       7.2                                                   ______________________________________                                    

EXAMPLE 22 IMMUNIZATION WITH VCP-16 TO PROTECT MICE AGAINST CHALLENGEWITH LIVE RABIES VIRUS

Group of 20, four to six week old mice were inoculated in the footpadwith 50 to 100 ul of a range of dilutions of either of two recombinants:(a) vFP-6- the fowlpox-rabies recombinant described in Example 6, and(b) vCP-16- the canarypox-rabies recombinant described in Example 13.

At 14 days, 10 mice from each group were sacrificed and the serumcollected. The anti-rabies titer in the serum was calculated using anRFFI test previously described in Example 7. The remaining 10 mice ineach group were challenged by intracerebral inoculation with the CVSstrain of rabies virus used in Example 7. Each mouse received 30 ulcorresponding to 16 mouse LD₅₀. At 28 days, surviving mice were assessedand the protective dose 50 (PD₅₀) calculated. The results are shown inTable XIV.

The level of protection of mice found by inoculation of vFP-6 confirmsthe result found on inoculation of the fowlpox recombinant vFP-3discussed in Example 7. The level of protection afforded by inoculationof vCP-16 is considerably higher. On the basis of the calculated PD₅₀the canarypox-rabies recombinant is 100 times more effective inprotection against rabies challenge than is the fowlpox-rabiesrecombinant.

                  TABLE XIV                                                       ______________________________________                                        Protective Immunity to Rabies Virus Challenge                                 Elicited Two Avipox-Rabies Recombinants                                       Fowlpox vFP-6     Canarypox vCP-16                                            Inoculum                                                                              RFFI    Survival  Inoculum                                                                             RFFI   Survival                              Dose    Titer   Ratio     Dose   Titer  Ratio                                 ______________________________________                                        .sup. 7.5.sup.a                                                                       .sup. 2.3.sup.b                                                                       7/10      6.5    2.5    10/10                                 5.5     1.8     5/10      4.5    1.9    8/10                                  3.5     0.7     0/10      2.5    1.1    1/10                                  1.5     0.6     0/10      0.5    0.4    0/10                                  1 PD.sub.50 = 6.17                                                                              1 PD.sub.50 = 4.18                                          ______________________________________                                         .sup.a Virus titers expressed as log.sub.10 TCID.sub.50                       .sup.b RFFI titer expressed as log.sub.10 of highest serum dilution givin     greater than 50% reduction in the number of fluorescing wells in an RFFI      test.                                                                    

EXAMPLE 23 USE OF FOWLPOX PROMOTER ELEMENTS TO EXPRESS FOREIGN GENES

I. Identification of the fowlpox gene encoding a 25.8 kilodaltons (KD)gene product. Visualization of protein species present in fowlpox (FP-1)infected CEF lysates by Coomassie brilliant blue staining ofSDS-polyacrylamide gels revealed an abundant species with an apparentmolecular weight of 25.8 KD. This protein was not present in uninfectedcell lysates. Pulse-experiments using ³⁵ S-methionine to radiolabelsynthesized proteins at specific times post infection again demonstratedthe abundance of the FP-1 induced protein and showed that it issynthesized from 6 hours to 54 hours postinfection. At its peak levelthis FP-1 25.8 KD protein accounts for approximately 5% to 10% of totalprotein present in the cell lysate.

The abundance of the FP-1 induced 25.8 KD protein suggested that thegene encoding this gene product is regulated by a strong FP-1 promoterelement. In order to localize this promoter element for subsequent usein the expression of foreign genes is poxvirus recombinants, a polysomepreparation was obtained from FP-1 infected CEF cells at 54 hourspostinfection. RNA was isolated from this polysome preparation and whenused to program a rabbit reticulocyte in vitro translation systemgenerated predominantly the 25.8 KD FP-1 protein.

The polysome RNA was also used as a template for first strand cDNAsynthesis using oligo (dT) 12-18 as a primer. The first strand cDNA wasused as a hybridization probe in Southern blot analyses with FP-1genomic digests. Results from these hybridization analyses suggestedthat the gene encoding the 25.8 KD protein was contained in a 10.5 KbpHind III fragment. This genomic Hind III fragment was subsequentlyisolated and ligated into a commercial vector, pBS (Stratagene, LaJolla, Calif.), and the clone was designated pFP23k-1. Furtherhybridization analyses using the first strand cDNA to probe digests ofpFP23k-1 localized the 25.8 Kd gene to a 3.2 Kbp Eco RV sub-fragment.The fragment was subcloned into pBS and designated pFP23k-2.

Approximately 2.4 Kbp of this FP-1 Eco RV fragment has been sequenced bythe Sanger dideoxy chain termination method (Sanger et al., Proc. Natl.Acad. Sci. USA 74, 5463-5467 [1977]). Analysis of the sequence revealsan open reading frame (ORF) which encodes a gene product with amolecular weight of 25.8 KD. In vitro run-off transcription of this ORFby bacteriophage T7 polymerase (Stratagene, La Jolla, Calif.) in a pBSvector generates an RNA species which when used to program a rabbitreticulocyte in vitro translation system (Promega Biotec, Madison, Wis.)yields a polypeptide species with an apparent molecular weight of 25.8KD. This polypeptide comigrates with the abundant 25.8 KD proteinobserved in lysates from FP-1 infected CEFs on an SDS-polyacrylamidegel. These results suggest that this is the gene encoding the abundantFP-1 induced 25.8 KD gene product.

II. Use of the upstream promoter elements of the FP-1 25.8 KD gene toexpress Feline Leukemia virus (FeLV) env ene in FP-1 and vacciniarecombinants. A 270 bp Eco RV/Eco RI fragment containing the FP-1 25.8KD gene regulatory region (FP25.8K promoter) and 21 bp of the 25.8 KDgene coding sequence was isolated from pFP23k-2. Below is presented thenucleotide sequence of the FP 25.8K promoter region used to derivepFeLV25.8F1 and pFeLV25.81A. This 270 nucleotide sequence provides 249nucleotides of the region upstream of the initiation codon (ATG) for the25.8 KD gene product and the first 21 bp of the coding sequence.##STR6##

This fragment was blunt-ended and then inserted into a Sma I digestedFP-1 insertion vector (pFeLVF1; see Example 15) containing the FeLV envsequences. This insertion vector enabled recombination with the f7 locusof the FP-1 genome. Insertion of the FP25.8K promoter upstream sequences5' to the FeLV env gene and in the proper orientation was confirmed bysequence analysis. This insertion does not provide a perfect ATG for ATGsubstitution but the ATG provided by the 25.8 KD gene is out of framewith the FeLV env ATG, so no fusion protein is formed. The FP-1insertion plasmid containing the FP25.8 KD promoter upstream from theFeLV env gene was designated pFeLV25.8F1.

A similar construct was prepared using the vaccinia virus insertionvector, pFeLV1A, harboring the FeLV gene (see Example 15). The H6promoter was excised from pFeLV1A by digestion with Bgl II and Sma I.Following blunt-ending of the Bgl II restriction site, the blunt ended270 bp Eco RV/Eco RI fragment containing the FP25.8K promoter wasinserted juxtaposed 5' to the FeLV env gene. This construct wasconfirmed by sequence analysis. There is not a perfect ATG for ATGsubstitution in this recombinant either but the ATG from the 25.8 KDgene is not in frame with the ATG from the FeLV gene. The vaccinia(Copenhagen strain) insertion vector harboring the 25.8 KD gene upstreamregion juxtaposed 5' to the FeLV gene was designated pFeLV25.81A.

The insertion plasmids, pFeLV25.8F1 and pFeLV25.81A, were used for invitro recombination with FP-1 (pFeLV25.8F1) and the Copenhagen strain ofvaccinia virus (pFeLV25.81A) as the rescuing viruses. Progeny of therecombination were plated on appropriate cell monolayers and recombinantvirus selected by a beta-galactosidase linked Protein A Immunoscreen anda bovine anti-FeLV serum (Antibodies, Inc., Davis, Calif.). Preliminaryresults suggest that the FP25.8K promoter can regulate the expression offoreign genes in poxvirus recombinants.

EXAMPLE 24 SAFETY AND EFFICACY OF VFP-6 AND VCP-16 IN POULTRY

The two avipox recombinants vFP-6 and vCP-16 (described in Examples 6and 13) were inoculated into 18 day old chicken embryos, 1 day oldchickens and 28 day old chickens and the response of the birds evaluatedon 3 criteria 1) effects of vaccination on hatchability, vaccinalreactions and mortality 2) the immune response induced to the rabiesglycoprotein and 3) the immune response induced to fowlpox antigens. Theexperiments performed are described below.

A. Safety Tests. Groups of twenty 18 day old embryos were inoculatedinto the allantoic cavity with 3.0 or 4.0 log₁₀ TCID₅₀ of either vFP-6or vCP-16. After hatching the chickens were observed for 14 days whenthey were individually bled and the sera collected. The two recombinantsinoculated in the chicken embryos had no effect on the hatchability ofthe eggs and the chickens remained healthy during the 14 day observationperiod.

Groups of 10 SPF 1 day old chickens were inoculated with 3.0 log₁₀TCID₅₀ of each of the recombinants by the intra-muscular route. Thechickens were observed for 28 days and serum samples collected at 14 and28 days post-inoculation. No vaccinal reaction was seen at theinoculation site with either of the recombinants and the chickensremained healthy through the 28 day observation period.

Groups of ten 28 day old chickens were inoculated with each of therecombinant viruses receiving either 3.0 log₁₀ TCID₅₀ by theintra-muscular route or 3.0 log₁₀ TCID₅₀ by the cutaneous (wing web)route. Chickens were observed for 28 days and serum samples collected at14 and 28 days post inoculation. No reaction was seen afterintramuscular inoculation with either of the recombinants. Cutaneousinoculation resulted in a very small vaccinal reaction to fowlpox withlesions being heterogenous in size. Canarypox inoculation led to theproduction of a normal cutaneous lesion at the inoculation site. Alllesions had regressed by the end of the experiment.

B. Immune Response. The RFFI test previously described in Example 7 wasused to assess the antibody levels to the rabies glycoprotein. For eachgroup, the results were expressed with the geometric mean titer of theindividual serum converted to International Units (IU) according to astandard serum which contained 23.4 IU. The minimum positivity level wasfixed at one IU and was used to determine the percentage of positivebirds. Antibodies against the avipox viruses were tested with an ELISAmethod using the fowlpox virus strain as an antigen. Each serum samplewas diluted at 1/20 and 1/80. A standard curve was constructed using apositive and negative sera. The minimum positivity level was calculatedwith the mean of the different values of the negative sera added withtwo standard deviations.

The results of serological surveys are shown in Table XV for vFP-6 andTable XVI for vCP-16.

A limited serological response was observed with embryos inoculated witheither vFP-6 or vCP-16 for both rabies and fowlpox antigens. The fowlpoxvector induced a serological response to both antigens in a greaternumber of birds than did canarypox but the response was stillheterogenous.

Chickens inoculated at 1-day old with vFP-6 had a good serologicalresponse with all birds being seropositive to rabies and fowlpoxantigens by 28 days post-inoculation. The response to vCP-16 inoculationwas much lower with 40% of birds being seropositive for rabiesglycoprotein at 28 days and 10% seropositive for avipox antigens.

Chickens inoculated with vFP-6 by the intramuscular route at 28 days oldshowed 100% seroconversion to both antigens by 14 days post-inoculation.Although the majority of birds also seroconverted after cutaneousinoculation, titers achieved were lower for both rabies and avipoxantigens. As previously, chickens inoculated both by the intramuscularand cutaneous route with vCP-16 showed a variable response with amaximum of 70% seroconversion to rabies by intramuscular inoculation.The low level of seroconversion for avipox antigens after canarypoxinoculation may reflect the degree of serological relatedness betweenthe viruses.

The results indicate both vFP-6 and vCP-16 to be safe for inoculation ofchickens of a range of ages. The fowlpox vector vFP-6 appears to be moreefficient in inducing an immune response in chickens. Significantly,however, both recombinant avipox viruses, fowlpox and canarypox, areshown to be useful for immunization in ovum.

                                      TABLE XV                                    __________________________________________________________________________    Immunologic Response Against Fowlpox/Rabies Glycoprotein (vFP-6) in           Chickens at Different Ages                                                                Time                                                                          After Antibodies                                                  Dose        Inoculation                                                                         Rabies Glycoprotein                                                                         Fowlpox                                       Groups                                                                              (TCID50)                                                                            (Days)                                                                              Mean IU titer                                                                        % birds/1 IU                                                                         mean Elisa OD                                                                         % positive                            __________________________________________________________________________    Embryos                                                                             10.sup.3                                                                            3 + 14                                                                              0.28    15%   0.125    54%                                  18 days old                                                                         10.sup.4                                                                            3 + 14                                                                              0.87    30%   0.129    46%                                  Chickens                                                                            10.sup.3                                                                            14    1.8     90%   0.109    70%                                  1 day old   28    4.2    100%   0.234   100%                                  IM route                                                                      Chickens                                                                            10.sup.3                                                                            14    3.7    100%   0.317   100%                                  28 day old  28    2.7    100%   0.378   100%                                  IM route                                                                      Chickens                                                                            10.sup.3                                                                            14    1.6    100%   0.191   100%                                  28 day old  28    0.54    90%   0.161    80%                                  transfixion                                                                   route                                                                         __________________________________________________________________________

                                      TABLE XVI                                   __________________________________________________________________________    Immunologic Response Against Canarypox/Rabies Glycoprotein (vCP-16) in        Chickens at Different Ages                                                                Time                                                                          After Antibodies                                                  Dose        Inoculation                                                                         Rabies Glycoprotein                                                                         Canarypox                                     Groups                                                                              (TCID60)                                                                            (Days)                                                                              Mean IU titer                                                                        % birds/1 IU                                                                         mean Elisa OD                                                                         % positive                            __________________________________________________________________________    Embryos                                                                             10.sup.3                                                                            3 + 14                                                                              0.14    0%    0.068   25%                                   18 days old                                                                         10.sup.4                                                                            3 + 14                                                                              0.19    8%    0.059   25%                                   Chickens                                                                            10.sup.3                                                                            14    0.18   10%    0.027    0%                                   1 day old   28    0.21   40%    0.059   10%                                   IM route                                                                      Chickens                                                                            10.sup.3                                                                            14    0.61   70%    0.093   60%                                   28 day old  28    0.24   30%    0.087   30%                                   IM route                                                                      Chickens                                                                            10.sup.3                                                                            14    0.34   40%    0.071   30%                                   28 day old  28    0.11   10%    0.061   10%                                   transfixion                                                                   route                                                                         __________________________________________________________________________

EXAMPLE 25 SAFETY AND IMMUNOGENICITY OF vFP-6 INOCULATION OF PIGLETS

Two groups of three piglets were inoculated with the recombinant vFP-6by one of two routes:

a) three animals received 8.1 log₁₀ TCID₅₀ by intramuscular inoculation;and

b) three animals received the same dose by oral inoculation.

All animals were bled at weekly intervals and received a boosterinoculation of the same dose by the same route on day 35. Piglets wereobserved daily for clinical signs. Sera were tested for antifowlpoxantibodies by an ELISA test and a serum neutralization test. Rabiesantibodies were assayed in an RFFI test.

All piglets remained in good health and no lesions were observed afterinoculation. Temperature curves were normal with no difference beingapparent between inoculated and uninoculated animals.

Piglets inoculated both by the intramuscular route and oral routedeveloped a serological response to fowlpox antigens as measured byELISA and serum neutralization. A secondary response was evident afterthe booster inoculation (results not shown). All piglets also developedan immunological response to rabies glycoprotein as measured in an RFFItest and a booster effect is evident by both routes. These results areshown in Table XVII.

The results indicate that inoculation of a fowlpox/rabies recombinant isinnocuous in piglets and that the recombinant is able to produce asignificant immune response to the rabies glycoprotein by oral orintramuscular inoculation.

                  TABIE XVII                                                      ______________________________________                                        Antibody to Rabies Glycoprotein Produced in Piglets                           Inoculated with vFP-6                                                         Vaccination                                                                            Animal  Rabies Antibody on Days (RFFI Titer)                         Route    No.     14     21    28   35.sup.b                                                                           42    49                              ______________________________________                                        I.M.     984     2.4.sup.a                                                                            2.2   2.1  2.2  3     3                                        985     2.5    2.7   2.6  2.4  3     3                                        986     2.2    2.0   2.1  2.3  3     3                               Oral     987     3      2     2.1  2    3     3                                        988     2.9    2.4   2.2  2.4  2.7   2.5                                      989     2.8    2     1.7  1.8  2.4   2.5                             ______________________________________                                         .sup.a Titer expressed as log.sub.10 of highest serum dilution giving         greater than 50% reduction in the number of fluorescing wells in an RFFI      test                                                                          .sup.b Animals received second inoculation on day 35                     

What is claimed is:
 1. A method for inducing an immunological responsein a mammal to a mammalian pathogen, which method comprises inoculatingthe mammal with a recombinant avipox virus comprising DNA which codesfor and expresses an antigen of the pathogen adequate to producerecoverable antibody without productive replication of the virus in themammal.
 2. A method as in claim 1 wherein said avipox virus is selectedfrom the group consisting of fowlpox virus and canary pox virus.
 3. Amethod as in claim 1 wherein the antigen is selected from the groupconsisting of rabies G antigen, gp51,30 envelope antigen of bovineleukemia virus, FeLV envelope antigen of feline leukemia virus andglycoprotein D antigen of herpes simplex virus.
 4. A method as in claim1 wherein the DNA codes for and expresses an antigen of a pathogen of amammal selected from the group consisting of dogs, cats, mice, rabbits,cattle, sheep and pigs.
 5. A method as in claim 1 wherein the vertebrateis inoculated by introducing the virus into the vertebratesubcutaneously, intradermally, intramuscularly, or orally.
 6. Arecombinant avipox virus containing therein DNA from a non-avipox sourcewhich codes for an antigen of a mammalian pathogen in a nonessentialregion of the avipox genome.
 7. An immunological composition which, whenintroduced into a mammalian host, induces an immunological response inthat mammal to a given pathogen, wherein the composition comprises arecombinant avipox virus comprising DNA which codes for and expresses anantigen of said pathogen without productive replication of the virus inthe mammal.
 8. An immunological composition according to claim 7 whereinthe recombinant avipox virus is a recombinant fowlpox or canary poxvirus.
 9. An immunological composition according to claim 7, wherein theDNA codes for the rabies G protein and the composition is a vaccine. 10.An immunological composition according to any one of claims 7 or 8wherein the antigen is selected from the group consisting of rabies Gantigen, gp51,30 envelope antigen of bovine leukemia virus, FeLVenvelope antigen of feline leukemia virus and glycoprotein D antigen ofherpes simplex virus.
 11. A recombinant avipox virus which induces animmunological response to a mammalian pathogen when said virus isintroduced into a mammal, said recombinant avipox virus comprising DNAwhich codes for and expresses an antigen to said pathogen withoutproductive replication of the virus in the mammal.
 12. A recombinantavipox virus according to claim 11 wherein the DNA codes for anexpresses an antigen of a pathogen of a mammal selected from the groupconsisting of dogs, cats, mice, rabbits, cattle, sheep and pigs.
 13. Amethod for inducing an immunological response in an avian host to anon-avipox avian pathogen, which method comprises inoculating the avianhost with a recombinant avipox virus comprising DNA which codes for andexpresses an antigen adequately to produce recoverable antibodies of thepathogen.
 14. A method as in claim 13 wherein the avian host isinoculated by introducing the virus into the avian host subcutaneously,intradermally, intramuscularly, orally or in ovum.
 15. The method of anyone of claims 13 wherein the DNA codes for an antigen selected from thegroup consisting of an antigen of avian influenza, an antigen ofNewcastle Disease virus, an envelope antigen of rous associated virus,and an antigen of infectious bronchitis virus.
 16. The method of claim15 wherein the DNA codes for an antigen selected from the groupconsisting of avian influenza hemagglutinin antigen, a fusion proteinantigen of Newcastle Disease virus, an RAV-1 envelope antigen of rousassociated virus, a nucleoprotein antigen of avian influenza virus, amatrix antigen of infectious bronchitis virus and a preplomer antigen ofinfectious bronchitis virus.
 17. The method of any one of claims 13wherein the avian host is a turkey or a chicken.
 18. A recombinantavipox virus containing therein DNA from a non-avipox source which codesfor an antigen of an avian pathogen in a nonessential region of theavipox genome.
 19. A recombinant avipox virus as claimed in claim 18wherein the DNA is downstream from a suitable promoter.
 20. Arecombinant avipox virus as in claim 19 wherein the promoter is avaccinia promoter or an entomopox promoter.
 21. A recombinant avipoxvirus as in claim 20 wherein the promoter is a vaccinia promoterselected from the group consisting of HH, 11K, and Pi.
 22. A recombinantavipox virus as in claim 20 wherein the promoter is a 42K entomopoxpromoter.
 23. The recombinant avipox virus as in claim 18 wherein theDNA codes for an antigen selected from the group consisting of anantigen of avian influenza, an antigen of Newcastle Disease virus, anenvelope antigen of rous associated virus, and an antigen of infectiousbronchitis virus.
 24. The recombinant avipox virus as in claim 23wherein the DNA codes for an antigen selected from the group consistingof avian influenza hemagglutinin antigen, a fusion protein antigen ofNewcastle Disease virus, an RAV-1 envelope antigen of rous associatedvirus, a nucleoprotein antigen of avian influenza virus, a matrixantigen of infectious bronchitis virus and a preplomer antigen ofinfectious bronchitis virus.
 25. A recombinant avipox virus whichinduces an immunological response to an avian pathogen when said avipoxvirus is introduced into a bird, said recombinant avipox viruscomprising DNA which codes for and expresses an antigen adequately toproduce recoverable antibodies to said pathogen.
 26. A recombinant virusaccording to claim 25 wherein the antigen encoded by the DNA is selectedfrom the group consisting of avian influenza hemagglutinin antigen, afusion protein antigen of Newcastle disease virus, an RAV-1 envelopeantigen of rous associated virus, a nucleoprotein antigen of avianinfluenza virus, a matrix antigen of infectious bronchitis virus and apeplomer antigen of infectious bronchitis virus.
 27. A recombinantavipox virus according to claim 25 wherein the DNA is downstream from asuitable promoter sequence which is a vaccinia promoter or an entomopoxpromoter.
 28. A recombinant avipox virus according to claim 27 whereinthe promoter sequence is a vaccinia promoter sequence selected from thegroup consisting of HH, 11K and Pi sequences.
 29. A recombinant avipoxvirus according to claim 27 wherein the promoter sequence is a42entomopox promoter sequence.
 30. The recombinant avipox virus of anyone of the claims 18, 23, 24, 25 or 26 wherein the avipox virus iscanarypox virus or fowlpox virus.
 31. An immunological compositioncomprising a recombinant avipox virus according to any one of claims 18,23, 24, 25 or 26 in admixture with a suitable carrier or diluent.
 32. Animmunological composition comprising a recombinant avipox virusaccording to claim 30 in admixture with a suitable carrier or diluent.33. A recombinant avipox virus synthetically modified by the presence,in a nonessential region of the avipox genome, of DNA not naturallyoccurring in avipox virus.
 34. A method for expressing a gene product atrecoverable levels which comprises introducing into cells of an in vitrocell culture a recombinant avipox virus as claimed in claim 6, 18 or 33.35. The recombinant avipox virus as claimed in claim 18 wherein the DNAcodes for a Newcastle Disease virus antigen.
 36. An immunologicalcomposition comprising the recombinant avipox virus as claimed in claim35 and a suitable carrier as a diluent.
 37. The recombinant avipox virusof claim 6, 18 or 33 which expresses the DNA.